U.S. patent application number 12/640342 was filed with the patent office on 2010-04-29 for lipid nanoparticle based compositions and methods for the delivery of biologically active molecules.
Invention is credited to Tongqian Chen, Kurt Vagle, Chandra Vargeese, Weimin Wang, Ye Zhang.
Application Number | 20100105933 12/640342 |
Document ID | / |
Family ID | 39777042 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100105933 |
Kind Code |
A1 |
Chen; Tongqian ; et
al. |
April 29, 2010 |
LIPID NANOPARTICLE BASED COMPOSITIONS AND METHODS FOR THE DELIVERY
OF BIOLOGICALLY ACTIVE MOLECULES
Abstract
The present invention relates to novel cationic lipids,
transfection agents, microparticles, nanoparticles, and short
interfering nucleic acid (siNA) molecules. The invention also
features compositions, and methods of use for the study, diagnosis,
and treatment of traits, diseases and conditions that respond to
the modulation of gene expression and/or activity in a subject or
organism. Specifically, the invention relates to novel cationic
lipids, microparticles, nanoparticles and transfection agents that
effectively transfect or deliver biologically active molecules,
such as antibodies (e.g., monoclonal, chimeric, humanized etc.),
cholesterol, hormones, antivirals, peptides, proteins,
chemotherapeutics, small molecules, vitamins, co-factors,
nucleosides, nucleotides, oligonucleotides, enzymatic nucleic
acids, antisense nucleic acids, triplex forming oligonucleotides,
2,5-A chimeras, dsRNA, allozymes, aptamers, decoys and analogs
thereof, and small nucleic acid molecules, such as short
interfering nucleic acid (siNA), short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), and RNAi inhibitor molecules, to relevant cells and/or
tissues, such as in a subject or organism. Such novel cationic
lipids, microparticles, nanoparticles and transfection agents are
useful, for example, in providing compositions to prevent, inhibit,
or treat diseases, conditions, or traits in a cell, subject or
organism. The compositions described herein are generally referred
to as formulated molecular compositions (FMC) or lipid
nanoparticles (LNP).
Inventors: |
Chen; Tongqian; (Irvine,
CA) ; Vagle; Kurt; (Longmont, CO) ; Vargeese;
Chandra; (Schwenksville, PA) ; Wang; Weimin;
(Churchville, PA) ; Zhang; Ye; (Lower Gwynedd,
PA) |
Correspondence
Address: |
MERCK
P O BOX 2000
RAHWAY
NJ
07065-0907
US
|
Family ID: |
39777042 |
Appl. No.: |
12/640342 |
Filed: |
December 17, 2009 |
Related U.S. Patent Documents
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Application
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12027952 |
Feb 7, 2008 |
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12640342 |
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11586102 |
Oct 24, 2006 |
7404969 |
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12027952 |
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11353630 |
Feb 14, 2006 |
7514099 |
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11586102 |
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60652787 |
Feb 14, 2005 |
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60678531 |
May 6, 2005 |
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60703946 |
Jul 29, 2005 |
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60737024 |
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Current U.S.
Class: |
552/544 |
Current CPC
Class: |
C07C 217/28 20130101;
A61P 31/18 20180101; C12N 15/88 20130101; A61P 25/08 20180101; A61P
25/28 20180101; A61P 33/02 20180101; A61P 9/08 20180101; A61P 37/06
20180101; A61P 1/16 20180101; A61P 9/12 20180101; A61P 31/06
20180101; A61P 13/02 20180101; Y10S 977/797 20130101; A61P 17/00
20180101; A61P 27/16 20180101; A61P 29/00 20180101; A61P 9/02
20180101; A61P 19/02 20180101; A61K 47/6911 20170801; A61P 31/04
20180101; A61P 19/08 20180101; A61P 27/10 20180101; A61P 13/12
20180101; A61P 25/06 20180101; A61P 35/02 20180101; A61K 9/1271
20130101; A61P 1/04 20180101; A61P 37/08 20180101; A61P 9/06
20180101; A61P 25/10 20180101; A61P 25/16 20180101; A61P 43/00
20180101; A61P 9/00 20180101; A61P 11/06 20180101; A61P 21/00
20180101; A61P 5/40 20180101; A61P 9/14 20180101; A61P 11/02
20180101; A61P 3/10 20180101; A61P 9/10 20180101; A61P 21/04
20180101; A61P 31/16 20180101; A61P 33/14 20180101; A61P 3/06
20180101; A61P 25/02 20180101; A61P 31/00 20180101; A61P 31/08
20180101; A61P 31/22 20180101; A61P 1/14 20180101; A61P 17/02
20180101; A61P 1/08 20180101; A61P 27/14 20180101; A61P 9/04
20180101; A61P 27/06 20180101; A61P 37/02 20180101; A61P 27/02
20180101; A61P 31/12 20180101; A61P 11/00 20180101; A61P 17/06
20180101; A61P 3/04 20180101; A61P 5/14 20180101; A61P 25/00
20180101; A61P 35/00 20180101; A61P 7/06 20180101; A61P 25/14
20180101; A61P 31/10 20180101 |
Class at
Publication: |
552/544 |
International
Class: |
C07J 9/00 20060101
C07J009/00 |
Claims
1-11. (canceled)
12. A compound having Formula CLI: ##STR00079## wherein each R1 and
R2 is methyl; R3 is a C9-C24 aliphatic saturated or unsaturated
hydrocarbon, L comprises a C1 to C10 alkyl linker, and R4 comprises
cholesterol.
13. A compound having Formula: ##STR00080##
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/353,630, filed Feb. 14, 2006, which claims
the benefit of U.S. Provisional patent application No. 60/652,787,
filed Feb. 14, 2005, U.S. Provisional patent application No.
60/678,531, filed May 6, 2005, U.S. Provisional patent application
No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent
application No. 60/737,024, filed Nov. 15, 2005. These applications
are incorporated by reference herein in their entirety including
the drawings.
FIELD OF THE INVENTION
[0002] The present invention relates to novel particle forming
delivery agents including cationic lipids, microparticles, and
nanoparticles that are useful for delivering various molecules to
cells. The invention also features compositions, and methods of use
for the study, diagnosis, and treatment of traits, diseases and
conditions that respond to the modulation of gene expression and/or
activity in a subject or organism. Specifically, the invention
relates to novel cationic lipids, microparticles, nanoparticles and
transfection agents that effectively transfect or deliver
biologically active molecules, such as antibodies (e.g.,
monoclonal, chimeric, humanized etc.), cholesterol, hormones,
antivirals, peptides, proteins, chemotherapeutics, small molecules,
vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,
enzymatic nucleic acids, antisense nucleic acids, triplex forming
oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and
analogs thereof, and small nucleic acid molecules, such as short
interfering nucleic acid (siNA), short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin
RNA (shRNA) molecules, to relevant cells and/or tissues, such as in
a subject or organism. Such novel cationic lipids, microparticles,
nanoparticles and transfection agents are useful, for example, in
providing compositions to prevent, inhibit, or treat diseases,
conditions, or traits in a cell, subject or organism.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to the delivery of
biologically active molecules to cells. Specifically, the invention
relates to compounds, compositions and methods for delivering
nucleic acids, polynucleotides, and oligonucleotides such RNA, DNA
and analogs thereof, peptides, polypeptides, proteins, antibodies,
hormones and small molecules, to cells by facilitating transport
across cellular membranes in, for example, epithelial tissues and
endothelial tissues. The compounds, compositions and methods of the
invention are useful in therapeutic, research, and diagnostic
applications that rely upon the efficient transfer of biologically
active molecules into cells, tissues, and organs. The discussion is
provided only for understanding of the invention that follows. This
summary is not an admission that any of the work described below is
prior art to the claimed invention.
[0004] The cellular delivery of various therapeutic compounds, such
as antiviral and chemotherapeutic agents, is usually compromised by
two limitations. First the selectivity of a number of therapeutic
agents is often low, resulting in high toxicity to normal tissues.
Secondly, the trafficking of many compounds into living cells is
highly restricted by the complex membrane systems of the cell.
Specific transporters allow the selective entry of nutrients or
regulatory molecules, while excluding most exogenous molecules such
as nucleic acids and proteins. Various strategies can be used to
improve transport of compounds into cells, including the use of
lipid carriers, biodegradable polymers, and various conjugate
systems.
[0005] The most well studied approaches for improving the transport
of foreign nucleic acids into cells involve the use of viral
vectors or cationic lipids and related cytofectins. Viral vectors
can be used to transfer genes efficiently into some cell types, but
they generally cannot be used to introduce chemically synthesized
molecules into cells. An alternative approach is to use delivery
formulations incorporating cationic lipids, which interact with
nucleic acids through one end and lipids or membrane systems
through another (for a review see Feigner, 1990, Advanced Drug
Delivery Reviews, 5,162-187; Feigner 1993, J. Liposome Res.,
3,3-16). Synthetic nucleic acids as well as plasmids can be
delivered using the cytofectins, although the utility of such
compounds is often limited by cell-type specificity, requirement
for low serum during transfection, and toxicity.
[0006] Another approach to delivering biologically active molecules
involves the use of conjugates. Conjugates are often selected based
on the ability of certain molecules to be selectively transported
into specific cells, for example via receptor-mediated endocytosis.
By attaching a compound of interest to molecules that are actively
transported across the cellular membranes, the effective transfer
of that compound into cells or specific cellular organelles can be
realized. Alternately, molecules that are able to penetrate
cellular membranes without active transport mechanisms, for
example, various lipophilic molecules, can be used to deliver
compounds of interest. Examples of molecules that can be utilized
as conjugates include but are not limited to peptides, hormones,
fatty acids, vitamins, flavonoids, sugars, reporter molecules,
reporter enzymes, chelators, porphyrins, intercalcators, and other
molecules that are capable of penetrating cellular membranes,
either by active transport or passive transport.
[0007] The delivery of compounds to specific cell types, for
example, cancer cells or cells specific to particular tissues and
organs, can be accomplished by utilizing receptors associated with
specific cell types. Particular receptors are overexpressed in
certain cancerous cells, including the high affinity folic acid
receptor. For example, the high affinity folate receptor is a tumor
marker that is overexpressed in a variety of neoplastic tissues,
including breast, ovarian, cervical, colorectal, renal, and
nasoparyngeal tumors, but is expressed to a very limited extent in
normal tissues. The use of folic acid based conjugates to transport
exogenous compounds across cell membranes can provide a targeted
delivery approach to the treatment and diagnosis of disease and can
provide a reduction in the required dose of therapeutic compounds.
Furthermore, therapeutic bioavailability, pharmacodynamics, and
pharmacokinetic parameters can be modulated through the use of
bioconjugates, including folate bioconjugates. Godwin et al., 1972,
J. Biol. Chem., 247, 2266-2271, report the synthesis of
biologically active pteroyloligo-L-glutamates. Habus et al., 1998,
Bioconjugate Chem., 9, 283-291, describe a method for the solid
phase synthesis of certain oligonucleotide-folate conjugates. Cook,
U.S. Pat. No. 6,721,208, describes certain oligonucleotides
modified with specific conjugate groups. The use of biotin and
folate conjugates to enhance transmembrane transport of exogenous
molecules, including specific oligonucleotides has been reported by
Low et al., U.S. Pat. Nos. 5,416,016, 5,108,921, and International
PCT publication No. WO 90/12096. Manoharan et al., International
PCT publication No. WO 99/66063 describe certain folate conjugates,
including specific nucleic acid folate conjugates with a
phosphoramidite moiety attached to the nucleic acid component of
the conjugate, and methods for the synthesis of these folate
conjugates. Nomura et al., 2000, J. Org. Chem., 65, 5016-5021,
describe the synthesis of an intermediate,
alpha-[2-(trimethylsilyl)ethoxycarbonyl]folic acid, useful in the
synthesis of ceratin types of folate-nucleoside conjugates. Guzaev
et al., U.S. Pat. No. 6,335,434, describes the synthesis of certain
folate oligonucleotide conjugates. Vargeese et al., International
PCT Publication No. WO 02/094185 and U.S. Patent Application
Publication Nos. 20030130186 and 20040110296 describe certain
nucleic acid conjugates.
[0008] The delivery of compounds to other cell types can be
accomplished by utilizing receptors associated with a certain type
of cell, such as hepatocytes. For example, drug delivery systems
utilizing receptor-mediated endocytosis have been employed to
achieve drug targeting as well as drug-uptake enhancement. The
asialoglycoprotein receptor (ASGPr) (see for example Wu and Wu,
1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and
binds branched galactose-terminal glycoproteins, such as
asialoorosomucoid (ASOR). Binding of such glycoproteins or
synthetic glycoconjugates to the receptor takes place with an
affinity that strongly depends on the degree of branching of the
oligosaccharide chain, for example, triatennary structures are
bound with greater affinity than biatenarry or monoatennary chains
(Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al.,
1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987,
Glycoconjugate J., 4, 317-328, obtained this high specificity
through the use of N-acetyl-D-galactosamine as the carbohydrate
moiety, which has higher affinity for the receptor, compared to
galactose. This "clustering effect" has also been described for the
binding and uptake of mannosyl-terminating glycoproteins or
glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24,
1388-1395). The use of galactose and galactosamine based conjugates
to transport exogenous compounds across cell membranes can provide
a targeted delivery approach to the treatment of liver disease such
as HBV and HCV infection or hepatocellular carcinoma. The use of
bioconjugates can also provide a reduction in the required dose of
therapeutic compounds required for treatment. Furthermore,
therapeutic bioavailability, pharmacodynamics, and pharmacokinetic
parameters can be modulated through the use of bioconjugates.
[0009] A number of peptide based cellular transporters have been
developed by several research groups. These peptides are capable of
crossing cellular membranes in vitro and in vivo with high
efficiency. Examples of such fusogenic peptides include a 16-amino
acid fragment of the homeodomain of ANTENNAPEDIA, a Drosophila
transcription factor (Wang et al., 1995, PNAS USA., 92, 3318-3322);
a 17-mer fragment representing the hydrophobic region of the signal
sequence of Kaposi fibroblast growth factor with or without NLS
domain (Antopolsky et al., 1999, Bioconj. Chem., 10, 598-606); a
17-mer signal peptide sequence of caiman crocodylus Ig(5) light
chain (Chaloin et al., 1997, Biochem. Biophys. Res. Comm., 243,
601-608); a 17-amino acid fusion sequence of HIV envelope
glycoprotein gp4114, (Morris et al.,1997, Nucleic Acids Res., 25,
2730-2736); the HIV-1 Tat49-57 fragment (Schwarze et al., 1999,
Science, 285, 1569-1572); a transportan A--achimeric 27-mer
consisting of N-terminal fragment of neuropeptide galanine and
membrane interacting wasp venom peptide mastoporan (Lindgren et
al., 2000, Bioconjugate Chem., 11, 619-626); and a 24-mer derived
from influenza virus hemagglutinin envelop glycoprotein (Bongartz
et al., 1994, Nucleic Acids Res., 22, 4681-4688). These peptides
were successfully used as part of an antisense
oligodeoxyribonucleotide-peptide conjugate for cell culture
transfection without lipids. In a number of cases, such conjugates
demonstrated better cell culture efficacy then parent
oligonucleotides transfected using lipid delivery. In addition, use
of phage display techniques has identified several organ targeting
and tumor targeting peptides in vivo (Ruoslahti, 1996, Ann. Rev.
Cell Dev. Biol., 12, 697-715). Conjugation of tumor targeting
peptides to doxorubicin has been shown to significantly improve the
toxicity profile and has demonstrated enhanced efficacy of
doxorubicin in the in vivo murine cancer model MDA-MB-435 breast
carcinoma (Arap et al., 1998, Science, 279, 377-380).
[0010] Another approach to the intracellular delivery of
biologically active molecules involves the use of cationic
polymers. For example, Ryser et al., International PCT Publication
No. WO 79/00515 describes the use of high molecular weight lysine
polymers for increasing the transport of various molecules across
cellular membranes. Rothbard et al., International PCT Publication
No. WO 98/52614, describes certain methods and compositions for
transporting drugs and macromolecules across biological membranes
in which the drug or macromolecule is covalently attached to a
transport polymer consisting of from 6 to 25 subunits, at least 50%
of which contain a guanidino or amidino side chain. The transport
polymers are preferably polyarginine peptides composed of all D-,
all L- or mixtures of D- and L-arginine. Rothbard et al., U.S.
Patent Application Publication No. 20030082356, describes certain
poly-lysine and poly-arginine compounds for the delivery of drugs
and other agents across epithelial tissues, including the skin,
gastrointestinal tract, pulmonary epithelium and blood brain
barrier. Wendel et al., U.S. Patent Application Publication No.
20030032593, describes certain polyarginine compounds. Rothbard et
al., U.S. Patent Application Publication No. 20030022831, describes
certain poly-lysine and poly-arginine compounds for intra-ocular
delivery of drugs. Kosak, U.S. Patent Application Publication No.
20010034333, describes certain cyclodextran polymers compositions
that include a cross-linked cationic polymer component. Beigelman
et al., U.S. Pat. No. 6,395,713; Reynolds et al., International PCT
Publication No. WO 99/04819; Beigelman et al., International PCT
Publication No. WO 99/05094; and Beigelman et al., U.S. Patent
Application Publication No. 20030073640 describe certain lipid
based formulations.
[0011] Another approach to the intracellular delivery of
biologically active molecules involves the use of liposomes or
other particle forming compositions. Since the first description of
liposomes in 1965, by Bangham (J. Mol. Biol. 13, 238-252), there
has been a sustained interest and effort in the area of developing
lipid-based carrier systems for the delivery of pharmaceutically
active compounds. Liposomes are attractive drug carriers since they
protect biological molecules from degradation while improving their
cellular uptake. One of the most commonly used classes of liposome
formulations for delivering polyanions (e.g., DNA) is that which
contains cationic lipids. Lipid aggregates can be formed with
macromolecules using cationic lipids alone or including other
lipids and amphiphiles such as phosphatidylethanolamine. It is well
known in the art that both the composition of the lipid formulation
as well as its method of preparation have effect on the structure
and size of the resultant anionic macromolecule-cationic lipid
aggregate. These factors can be modulated to optimize delivery of
polyanions to specific cell types in vitro and in vivo. The use of
cationic lipids for cellular delivery of biologically active
molecules has several advantages. The encapsulation of anionic
compounds using cationic lipids is essentially quantitative due to
electrostatic interaction. In addition, it is believed that the
cationic lipids interact with the negatively charged cell membranes
initiating cellular membrane transport (Akhtar et al., 1992, Trends
Cell Bio., 2, 139; Xu et al., 1996, Biochemistry 35, 5616).
[0012] Experiments have shown that plasmid DNA can be encapsulated
in small particles that consist of a single plasmid encapsulated
within a bilayer lipid vesicle (Wheeler, et al., 1999, Gene Therapy
6, 271-281). These particles typically contain the fusogenic lipid
dioleoylphosphatidylethanolamine (DOPE), low levels of a cationic
lipid, and can be stabilized in aqueous media by the presence of a
poly(ethylene glycol) (PEG) coating. These particles have systemic
applications as they exhibit extended circulation lifetimes
following intravenous (i.v.) injection, can accumulate
preferentially in various tissues and organs or tumors due to the
enhanced vascular permeability in such regions, and can be designed
to escape the lyosomic pathway of endocytosis by disruption of
endosomal membranes. These properties can be useful in delivering
biologically active molecules to various cell types for
experimental and therapeutic applications. For example, the
effective use of nucleic acid technologies such as short
interfering RNA (siRNA), antisense, ribozymes, decoys, triplex
forming oligonucleotides, 2-5A oligonucleotides, and aptamers in
vitro and in vivo may benefit from efficient delivery of these
compounds across cellular membranes. Lewis et al., U.S. Patent
Application Publication No. 20030125281, describes certain
compositions consisting of the combination of siRNA, certain
amphipathic compounds, and certain polycations. MacLachlan, U.S.
Patent Application Publication No. 20030077829, describes certain
lipid based formulations. MacLachlan, International PCT Publication
No. WO 05/007196, describes certain lipid encapsulated interfering
RNA formulations. Vargeese et al., International PCT Publication
No. WO2005007854 describes certain polycationic compositions for
the cellular delivery of polynucleotides. McSwiggen et al.,
International PCT Publication Nos. WO 05/019453, WO 03/70918, WO
03/74654 and U.S. Patent Application Publication Nos. 20050020525
and 20050032733, describes short interfering nucleic acid molecules
(siNA) and various technologies for the delivery of siNA molecules
and other polynucleotides.
[0013] In addition, recent work involving cationic lipid particles
demonstrated the formation of two structurally different complexes
comprising nucleic acid (or other polyanionic compound) and
cationic lipid (Safinya et al., Science, 281: 78-81 (1998). One
structure comprises a multilamellar structure with nucleic acid
monolayers sandwiched between cationic lipid bilayers ("lamellar
structure") (FIG. 7). A second structure comprises a two
dimensional hexagonal columnar phase structure ("inverted hexagonal
structure") in which nucleic acid molecules are encircled by
cationic lipid in the formation of a hexagonal structure (FIG. 7).
Safinya et al. demonstrated that the inverted hexagonal structure
transfects mammalian cells more efficiently than the lamellar
structure. Further, optical microscopy studies showed that the
complexes comprising the lamellar structure bind stably to anionic
vesicles without fusing to the vesicles, whereas the complexes
comprising the inverted hexagonal structure are unstable and
rapidly fuse to the anionic vesicles, releasing the nucleic acid
upon fusion.
[0014] The structural transformation from lamellar phase to
inverted hexagonal phase complexes is achieved either by
incorporating a suitable helper lipid that assists in the adoption
of an inverted hexagonal structure or by using a co-surfactant,
such as hexanol. However, neither of these transformation
conditions are suitable for delivery in biological systems.
Furthermore, while the inverted hexagonal complex exhibits greater
transfection efficiency, it has very poor serum stability compared
to the lamellar complex. Thus, there remains a need to design
delivery agents that are serum stable, i.e. stable in circulation,
that can undergo structural transformation, for example from
lamellar phase to inverse hexagonal phase, under biological
conditions.
[0015] The present application provides compounds, compositions and
methods for significantly improving the efficiency of systemic and
local delivery of biologically active molecules. Among other
things, the present application provides compounds, compositions
and methods for making and using novel delivery agents that are
stable in circulation and undergo structural changes under
appropriate physiological conditions (e.g., pH) which increase the
efficiency of delivery of biologically active molecules.
[0016] Various lipid nucleic acid particles and methods of
preparation thereof are described in U.S. Patent Application
Publication Nos. 20030077829, 20030108886, 20060051405,
20060083780, 20030104044, 20060051405, 20040142025, 200600837880,
20050064595, 2005/0175682, 2005/0118253, 20050255153 and
20050008689; and U.S. Pat. Nos. 5,885,613; 6,586,001; 6,858,225;
6,858,224; 6,815,432; 6,586,410; 6,534,484; and 6,287,591.
SUMMARY OF THE INVENTION
[0017] The present invention features compounds, compositions, and
methods to facilitate delivery of various molecules into a
biological system, such as cells. The compounds, compositions, and
methods provided by the instant invention can impart therapeutic
activity by transferring therapeutic compounds across cellular
membranes or across one or more layers of epithelial or endothelial
tissue. The present invention encompasses the design and synthesis
of novel agents for the delivery of molecules, including but not
limited to small molecules, lipids, nucleosides, nucleotides,
nucleic acids, polynucleotides, oligonucleotides, antibodies,
toxins, negatively charged polymers and other polymers, for example
proteins, peptides, hormones, carbohydrates, or polyamines, across
cellular membranes. Non-limiting examples of polynucleotides that
can be delivered across cellular membranes using the compounds and
methods of the invention include short interfering nucleic acids
(siNA) (which includes siRNAs), antisense oligonucleotides,
enzymatic nucleic acid molecules, 2',5'-oligoadenylates, triplex
forming oligonucleotides, aptamers, and decoys. In general, the
transporters described are designed to be used either individually
or as part of a multi-component system, with or without degradable
linkers. The compounds of the invention (generally shown in the
Formulae below), when formulated into compositions, are expected to
improve delivery of molecules into a number of cell types
originating from different tissues, in the presence or absence of
serum.
[0018] The compounds, compositions, and methods of the invention
are useful for delivering biologically active molecules (e.g.,
siNAs, siRNAs, miRNAs, siRNA and miRNA inhibitors, nucleic acids,
polynucleotides, oligonucleotides, peptides, polypeptides,
proteins, hormones, antibodies, and small molecules) to cells or
across epithelial and endothelial tissues, such as skin, mucous
membranes, vasculature tissues, gastrointestinal tissues, blood
brain barrier tissues, opthamological tissues, pulmonary tissues,
liver tissues, cardiac tissues, kidney tissues etc. The compounds,
compositions, and methods of the invention can be used both for
delivery to a particular site of administration or for systemic
delivery.
[0019] The compounds, compositions, and methods of the invention
can increase delivery or availability of biologically active
molecules (e.g., siNAs, siRNAs, miRNAs, siRNA and miRNA inhibitors,
nucleic acids, polynucleotides, oligonucleotides, peptides,
polypeptides, proteins, hormones, antibodies, and small molecules)
to cells or tissues compared to delivery of the molecules in the
absence of the compounds, compositions, and methods of the
invention. As such, the level of a biologically active molecule
inside a cell, tissue, or organism is increased in the presence of
the compounds and compositions of the invention compared to when
the compounds and compositions of the invention are absent.
[0020] In one aspect, the invention features novel cationic lipids,
transfection agents, microparticles, nanoparticles, and
formulations thereof with biologically active molecules. In another
embodiment, the invention features compositions, and methods of use
for the study, diagnosis, and treatment of traits, diseases, and
conditions that respond to the modulation of gene expression and/or
activity in a subject or organism. In another embodiment, the
invention features novel cationic lipids, microparticles,
nanoparticles transfection agents, and formulations that
effectively transfect or deliver small nucleic acid molecules, such
as short interfering nucleic acid (siNA), short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short
hairpin RNA (shRNA) molecules, and inibitors thereof (RNAi
inhibitors); to relevant cells and/or tissues, such as in a subject
or organism. Such novel cationic lipids, microparticles,
nanoparticles, transfection agents, and formulations are useful,
for example, in providing compositions to prevent, inhibit, or
treat diseases, conditions, or traits in a cell, subject or
organism as described herein.
[0021] In one aspect, the instant invention features various
cationic lipids, microparticles, nanoparticles, transfection
agents, and formulations for the delivery of chemically-modified
synthetic short interfering nucleic acid (siNA) molecules and/or
RNAi inhibitors that modulate target gene expression or activity in
cells, tissues, such as in a subject or organism, by RNA
interference (RNAi). The use of chemically-modified siNA improves
various properties of native siRNA molecules through increased
resistance to nuclease degradation in vivo, improved cellular
uptake, and improved pharmacokinetic properties in vivo. The
cationic lipids, microparticles, nanoparticles, transfection
agents, formulations, and siNA molecules and RNAi inhibitors of the
instant invention provide useful reagents and methods for a variety
of therapeutic, veterinary, diagnostic, target validation, genomic
discovery, genetic engineering, and pharmacogenomic
applications.
[0022] In one aspect, the invention features compositions and
methods that independently or in combination modulate the
expression of target genes encoding proteins, such as proteins
associated with the maintenance and/or development of a disease,
trait, or condition, such as a liver disease, trait, or condition.
These genes are referred to herein generally as target genes. Such
target genes are generally known in the art and transcripts of such
genes are commonly referenced by Genbank Accession Number, see for
example International PCT Publication No. WO 03/74654, serial No.
PCT/US03/05028, and U.S. patent application Ser. No. 10/923,536
both incorporated by reference herein). The description below of
the various aspects and embodiments of the invention is provided
with reference to exemplary target genes and target gene
transcripts. However, the various aspects and embodiments are also
directed to other target genes, such as gene homologs, gene
transcript variants, and gene polymorphisms (e.g., single
nucleotide polymorphism, (SNPs)) that are associated with certain
target genes. As such, the various aspects and embodiments are also
directed to other genes that are involved in pathways of signal
transduction or gene expression that are involved, for example, in
the maintenance and/or development of a disease, trait, or
condition. These additional genes can be analyzed for target sites
using the methods described for target genes herein. Thus, the
modulation of other genes and the effects of such modulation of the
other genes can be performed, determined, and measured as described
herein.
[0023] In one embodiment, the invention features a compound having
Formula CLI:
##STR00001##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (for example, monoester, diester), or succinyl
linker. In one embodiment, R1 and R2 are methyl, R3 is linoyl, L is
butyl, and R4 is cholesterol, which compound is generally referred
to herein as CLinDMA or
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-(cis,cis-9,1-
2-octadecadienoxy)propane.
[0024] In one embodiment, the invention features a compound having
Formula CLII:
##STR00002##
[0025] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester) or succinyl linker. In
one embodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, and
R4 is cholesterol.
[0026] In one embodiment, the invention features a compound having
Formula CLIII:
##STR00003##
[0027] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In one embodiment,
L is an acetal, amide, carbonyl, carbamide, carbamate, carbonate,
ester (i.e., monoester, diester), or succinyl linker. In one
embodiment, each R1 and R2 are methyl, R3 is linoyl, L is butyl,
and R4 is cholesterol.
[0028] In one embodiment, the invention features a compound having
Formula CLIV:
##STR00004##
[0029] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, each R1 and R2 are methyl, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0030] In one embodiment, the invention features a compound having
Formula CLV:
##STR00005##
[0031] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; and each R3 and R4 is independently a
C12-C24 aliphatic hydrocarbon, which can be the same or different.
In one embodiment, R1 and R2 each independently is methyl, ethyl,
propyl, isopropyl, or butyl. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, R1 and R2 are methyl, and R3 and R4 are
oleyl, this compound is generally referred to herein as DMOBA or
N,N-Dimethyl-3,4-dioleyloxybenzylamine.
[0032] In one embodiment, the invention features a compound having
Formula CLVI:
##STR00006##
[0033] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, and
R4 is cholesterol.
[0034] In one embodiment, the invention features a compound having
Formula CLVII:
##STR00007##
[0035] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R1 and R2 are methyl, R3 is linoyl, L is butyl, and
R4 is cholesterol.
[0036] In one embodiment, the invention features a compound having
Formula CLVIII:
##STR00008##
[0037] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; and each R3 and R4 is independently a
C12-C24 aliphatic hydrocarbon which can be the same or different.
In one embodiment, R1 and R2 each independently is methyl, ethyl,
propyl, isopropyl, or butyl. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each R1 and R2 are methyl, and R3 and
R4 are linoyl.
[0038] In one embodiment, the invention features a compound having
Formula CLIX:
##STR00009##
[0039] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamate carbamide,
carbamate, carbonate, ester (i.e., monoester, diester), or succinyl
linker. In one embodiment, each R1 and R2 are methyl, R3 is linoyl,
L is butyl, and R4 is cholesterol.
[0040] In one embodiment, the invention features a compound having
Formula CLX:
##STR00010##
[0041] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, each R1 and R2 are methyl, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0042] In one embodiment, the invention features a compound having
Formula CLXI:
##STR00011##
[0043] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, each R1 and R2 are methyl, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0044] In one embodiment, the invention features a compound having
Formula CLXIIa or CLXIIb:
##STR00012##
[0045] wherein R0 and each R1 and R2 is independently a C1 to C10
alkyl, alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic
saturated or unsaturated hydrocarbon, L is a linker, and R4 is
cholesterol, a cholesterol derivative, a steroid hormone, or a bile
acid. In one embodiment, R1 and R2 each independently is methyl,
ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 is
linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker. In
another embodiment, L is an acetal, amide, carbonyl, carbamide,
carbamate, carbonate, ester (i.e., monoester, diester), or succinyl
linker. In one embodiment, each R1 and R2 are methyl, R3 is linoyl,
L is butyl, and R4 is cholesterol.
[0046] In one embodiment, the invention features a compound having
Formula CLXIII:
##STR00013##
[0047] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 and R2 each independently is methyl, ethyl, propyl,
isopropyl, or butyl. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, each R1 and R2 are methyl, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0048] In one embodiment, the invention features a compound having
Formula CLXIVa and CLXIVb:
##STR00014##
[0049] wherein R0 and each R1 and R2 is independently a C1 to C10
alkyl, alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic
saturated or unsaturated hydrocarbon, L is a linker, and R4 is
cholesterol, a cholesterol derivative, a steroid hormone, or a bile
acid. In one embodiment, R1 and R2 each independently is methyl,
ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 is
linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker. In
another embodiment, L is an acetal, amide, carbonyl, carbamide,
carbamate, carbonate, ester (i.e., monoester, diester), or succinyl
linker. In one embodiment, each R1 and R2 are methyl, R3 is linoyl,
L is butyl, and R4 is cholesterol.
[0050] In one embodiment, the invention features a compound having
Formula CLXV:
##STR00015##
[0051] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; L is a linker, and each R3 is
independently cholesterol, a cholesterol derivative, a steroid
hormone, or a bile acid. In one embodiment, R1 and R2 each
independently is methyl, ethyl, propyl, isopropyl, or butyl. In one
embodiment, R3 is cholesterol. In one embodiment, L is a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker. In
another embodiment, L is an acetal, amide, carbonyl, carbamide,
carbamate, carbonate, ester (i.e., monoester, diester), or succinyl
linker. In one embodiment, each R1 and R2 are methyl, R3 is
cholesterol, and L is butyl.
[0052] In one embodiment, the invention features a compound having
Formula CLXVI:
##STR00016##
[0053] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; each L is a linker whose structure is
independent of the other L, and each R3 is independently
cholesterol, a cholesterol derivative, a steroid hormone, or a bile
acid. In one embodiment, R1 and R2 each independently is methyl,
ethyl, propyl, isopropyl, or butyl. In one embodiment, R3 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, each R1 and R2 are methyl, R3 is cholesterol, and L
is butyl.
[0054] In one embodiment, the invention features a compound having
Formula CLXVII:
##STR00017##
[0055] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon and R3 is a C9-C24 aliphatic saturated
or unsaturated hydrocarbon. In one embodiment, R1 and R2 each
independently is methyl, ethyl, propyl, isopropyl, or butyl. In one
embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each R1 and R2 are methyl and R3 is
linoyl.
[0056] In one embodiment, the invention features a compound having
Formula CLXVIII:
##STR00018##
[0057] wherein each R1 and R2 is independently a C1 to C10 alkyl,
alkynyl, or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon. In one embodiment, R1 and R2 each
independently is methyl, ethyl, propyl, isopropyl, or butyl. In one
embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each R1 and R2 are methyl and R3 is
linoyl.
[0058] In one embodiment, the invention features a compound having
Formula CLXIX:
##STR00019##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0059] In one embodiment, the invention features a compound having
Formula CLXX:
##STR00020##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0060] In one embodiment, the invention features a compound having
Formula CLXXI:
##STR00021##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0061] In one embodiment, the invention features a compound having
Formula CLXXII:
##STR00022##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0062] In one embodiment, the invention features a compound having
Formula CLXXIII:
##STR00023##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, and L is a linker. In one embodiment, R1
and R2 each independently is methyl, ethyl, propyl, isopropyl, or
butyl. In one embodiment, R3 and R4 each individually is linoyl,
isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,
arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, R3
or R4 is cholesterol, a cholesterol derivative, a steroid hormone,
or a bile acid. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl
linker.
[0063] In one embodiment, the invention features a compound having
Formula CLXXIV:
##STR00024##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, and L is a linker. In one embodiment, R1
and R2 each independently is methyl, ethyl, propyl, isopropyl, or
butyl. In one embodiment, R3 and R4 each individually is linoyl,
isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,
arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, R3
or R4 is cholesterol, a cholesterol derivative, a steroid hormone,
or a bile acid. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl
linker.
[0064] In one embodiment, the invention features a compound having
Formula CLXXV:
##STR00025##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, and L is a linker. In one embodiment, R1
and R2 each independently is methyl, ethyl, propyl, isopropyl, or
butyl. In one embodiment, R3 and R4 each individually is linoyl,
isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,
arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, R3
or R4 is cholesterol, a cholesterol derivative, a steroid hormone,
or a bile acid. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl
linker.
[0065] In one embodiment, the invention features a compound having
Formula CLXXVI:
##STR00026##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0066] In one embodiment, the invention features a compound having
Formula CLXXVII:
##STR00027##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, and L is a linker.
In one embodiment, R1 and R2 each independently is methyl, ethyl,
propyl, isopropyl, or butyl. In one embodiment, R3 and R4 each
individually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, R3 or R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment, L
is a C1 to C10 alkyl, alkyl ether, polyether, or polyethylene
glycol linker. In another embodiment, L is an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker.
[0067] In one embodiment, the invention features a compound having
Formula CLXXVIII:
##STR00028##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0068] In one embodiment, the invention features a compound having
Formula CLXXIX:
##STR00029##
wherein each R1 and R2 is independently a C1 to C10 alkyl, alkynyl,
or aryl hydrocarbon; R3 and R4 are each individually a C9-C24
aliphatic saturated or unsaturated hydrocarbon, which can be the
same or different. In one embodiment, R1 and R2 each independently
is methyl, ethyl, propyl, isopropyl, or butyl. In one embodiment,
R3 and R4 each individually is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R3 or R4 is cholesterol,
a cholesterol derivative, a steroid hormone, or a bile acid.
[0069] In one embodiment, the invention features a compound having
Formula CLXXX:
##STR00030##
wherein R3 is a C9-C24 aliphatic saturated or unsaturated
hydrocarbon, each L is independently a linker, and R4 is
cholesterol, a cholesterol derivative, a steroid hormone, or a bile
acid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R4 is cholesterol. In one
embodiment, each L is independently a C1 to C10 alkyl, alkyl ether,
polyether, or polyethylene glycol linker with or without a
disulphide linkage. In another embodiment, each L is independently
an acetal, amide, carbonyl, carbamide, carbamate, carbonate, ester
(for example, monoester, diester), or succinyl linker. In one
embodiment, R3 is linoyl and R4 is cholesterol.
[0070] In one embodiment, the invention features a compound having
Formula CLXXXI:
##STR00031##
[0071] wherein R3 is a C9-C24 aliphatic saturated or unsaturated
hydrocarbon, each L is independently a linker, and R4 is
cholesterol, a cholesterol derivative, a steroid hormone, or a bile
acid. In one embodiment, R3 is linoyl, isostearyl, oleyl, elaidyl,
petroselinyl, linolenyl, elaeostearyl, arachidyl, myristoyl,
palmitoyl, or lauroyl. In one embodiment, R4 is cholesterol. In one
embodiment, each L is independently a C1 to C10 alkyl, alkyl ether,
polyether, or polyethylene glycol linker with or without a
disulphide linkage. In another embodiment, each L is independently
an acetal, amide, carbonyl, carbamide, carbamate, carbonate, ester
(i.e., monoester, diester) or succinyl linker. In one embodiment,
R3 is linoyl, L is butyl, and R4 is cholesterol.
[0072] In one embodiment, the invention features a compound having
Formula CLXXXII:
##STR00032##
[0073] wherein each R1, R2 and R5 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
one embodiment, each L is independently an acetal, amide, carbonyl,
carbamide, carbamate, carbonate, ester (i.e., monoester, diester),
or succinyl linker. In one embodiment, R3 is linoyl and R4 is
cholesterol.
[0074] In one embodiment, the invention features a compound having
Formula CLXXXIII:
##STR00033##
[0075] wherein wherein each R1, R2 and R5 is independently
hydrogen, methyl, ethyl, propyl, isopropyl, or butyl, R3 is a
C9-C24 aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl and
R4 is cholesterol.
[0076] In one embodiment, the invention features a compound having
Formula CLXXXIV:
##STR00034##
[0077] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0078] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0079] In one embodiment, the invention features a compound having
Formula CLXXXV:
##STR00035##
[0080] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0081] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0082] In one embodiment, the invention features a compound having
Formula CLXXXVI:
##STR00036##
[0083] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0084] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0085] In one embodiment, the invention features a compound having
Formula CLXXXVII:
##STR00037##
[0086] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0087] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0088] In one embodiment, the invention features a compound having
Formula CLXXXVIII:
##STR00038##
[0089] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0090] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0091] In one embodiment, the invention features a compound having
Formula CLXXXIX:
##STR00039##
[0092] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0093] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0094] In one embodiment, the invention features a compound having
Formula CLXXXX:
##STR00040##
[0095] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0096] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0097] In one embodiment, the invention features a compound having
Formula CLXXXXI:
##STR00041##
[0098] wherein wherein each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, each R3 and R4 is
independently a C12-C24 aliphatic hydrocarbon, which can be the
same or different, and each L is independently a linker, which can
be present or absent. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, each L is independently a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker with
or without a disulphide linkage. In another embodiment, each L is
independently an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R3 and R4 are oleyl.
[0099] In one embodiment, each R1 and R2 is independently hydrogen,
methyl, ethyl, propyl, isopropyl, or butyl, R3 is a C9-C24
aliphatic saturated or unsaturated hydrocarbon, each L is
independently a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, each L is
independently a C1 to C10 alkyl, alkyl ether, polyether, or
polyethylene glycol linker with or without a disulphide linkage. In
another embodiment, each L is independently an acetal, amide,
carbonyl, carbamide, carbamate, carbonate, ester (i.e., monoester,
diester), or succinyl linker. In one embodiment, R3 is linoyl, L is
butyl, and R4 is cholesterol.
[0100] In one embodiment, the invention features a compound having
Formula CLXXXXII:
##STR00042##
[0101] wherein R3 is a C9-C24 aliphatic saturated or unsaturated
hydrocarbon, L is a linker, and R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
R3 is linoyl, isostearyl, oleyl, elaidyl, petroselinyl, linolenyl,
elaeostearyl, arachidyl, myristoyl, palmitoyl, or lauroyl. In one
embodiment, R4 is cholesterol. In one embodiment, L is a C1 to C10
alkyl, alkyl ether, polyether, or polyethylene glycol linker. In
another embodiment, L is an acetal, amide, carbonyl, carbamide,
carbamate, carbonate, ester (i.e., monoester, diester), or succinyl
linker. In one embodiment, R3 is linoyl, L is butyl, and R4 is
cholesterol.
[0102] In one embodiment, any of compounds CLI-CLXXXXII include a
biodegradable linkage as L, for example a disulphide linkage such
as:
##STR00043##
[0103] In one embodiment, the invention features a compound having
Formula NLI:
##STR00044##
wherein R1 is H, OH, or a C1 to C10 alkyl, alkynyl, or aryl
hydrocarbon or alcohol; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 is OH, methyl, ethyl, propyl, isopropyl, or butyl or
its corresponding alcohol. In one embodiment, R3 is linoyl,
isostearyl, oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl,
arachidyl, myristoyl, palmitoyl, or lauroyl. In one embodiment, R4
is cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (for example, monoester, diester), or succinyl
linker. In one embodiment, R1 is OH, R3 is linoyl, L is butyl, and
R4 is cholesterol.
[0104] In one embodiment, the invention features a compound having
Formula NLII:
##STR00045##
[0105] wherein R1 is H, OH, or a C1 to C10 alkyl, alkynyl, or aryl
hydrocarbon or alcohol; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 is methyl, ethyl, propyl, isopropyl, or butyl or its
corresponding alcohol. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester) or succinyl linker. In
one embodiment, R1 is OH, R3 is linoyl, L is butyl, and R4 is
cholesterol.
[0106] In one embodiment, the invention features a compound having
Formula NLIII:
##STR00046##
[0107] wherein R1 is H, OH, a C1 to C10 alkyl, alkynyl, or aryl
hydrocarbon or alcohol; and each R3 and R4 is independently a
C12-C24 aliphatic hydrocarbon, which can be the same or different.
In one embodiment, R1 is methyl, ethyl, propyl, isopropyl, or butyl
or its corresponding alcohol. In one embodiment, R3 and R4 each
independently is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, R1 is OH, and R3 and R4 are oleyl, this
compound is generally referred to herein as DOBA or
dioleyloxybenzyl alcohol.
[0108] In one embodiment, the invention features a compound having
Formula NLIV:
##STR00047##
[0109] wherein R1 is H, OH a C1 to C10 alkyl, alkynyl, or aryl
hydrocarbon or alcohol; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 is methyl, ethyl, propyl, isopropyl, or butyl or its
corresponding alcohol. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R1 is OH, R3 is linoyl, L is butyl, and R4 is
cholesterol.
[0110] In one embodiment, the invention features a compound having
Formula NLV:
##STR00048##
[0111] wherein R1 is H, OH a C1 to C10 alkyl, alkynyl, or aryl
hydrocarbon or alcohol; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, L is a linker, and R4 is cholesterol, a
cholesterol derivative, a steroid hormone, or a bile acid. In one
embodiment, R1 is methyl, ethyl, propyl, isopropyl, or butyl or its
corresponding alcohol. In one embodiment, R3 is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R4 is
cholesterol. In one embodiment, L is a C1 to C10 alkyl, alkyl
ether, polyether, or polyethylene glycol linker. In another
embodiment, L is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl linker. In
one embodiment, R1 is OH, R3 is linoyl, L is butyl, and R4 is
cholesterol.
[0112] In one embodiment, the invention features a compound having
Formula NLVI:
##STR00049##
wherein R1 is H, OH, a C1 to C10 alkyl, alkynyl, or aryl
hydrocarbon or alcohol; R3 is a C9-C24 aliphatic saturated or
unsaturated hydrocarbon, and each L is a linker. In one embodiment,
R1 is methyl, ethyl, propyl, isopropyl, or butyl or its
corresponding alcohol. In one embodiment, R3 and R4 each
individually is linoyl, isostearyl, oleyl, elaidyl, petroselinyl,
linolenyl, elaeostearyl, arachidyl, myristoyl, palmitoyl, or
lauroyl. In one embodiment, R3 or R4 is cholesterol, a cholesterol
derivative, a steroid hormone, or a bile acid. In one embodiment,
each L independently is a C1 to C10 alkyl, alkyl ether, polyether,
or polyethylene glycol linker. In another embodiment, each L
independently is an acetal, amide, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or succinyl
linker.
[0113] In one embodiment, the invention features a compound having
Formula NLVII:
##STR00050##
wherein R1 is independently H, OH, a C1 to C10 alkyl, alkynyl, or
aryl hydrocarbon or alcohol; R3 and R4 are each individually a
C9-C24 aliphatic saturated or unsaturated hydrocarbon, which can be
the same or different. In one embodiment, R1 is methyl, ethyl,
propyl, isopropyl, or butyl or its corresponding alcohol. In one
embodiment, R3 and R4 each individually is linoyl, isostearyl,
oleyl, elaidyl, petroselinyl, linolenyl, elaeostearyl, arachidyl,
myristoyl, palmitoyl, or lauroyl. In one embodiment, R3 or R4 is
cholesterol, a cholesterol derivative, a steroid hormone, or a bile
acid.
[0114] In one embodiment, each O--R3 and/or O--R4 of any compound
having Formulae CLI-CLXIV, CLXVII-CLXXII, CLXXVI, and
CLXXVIII-CLXXXIX further comprises a linker L (e.g., wherein
--O--R3 and/or --O--R4 as shown above is --O-L-R3 and/or --O-L-R4),
where L is a C1 to C10 alkyl, alkyl ether, polyether, polyethylene
glycol, acetal, amide, succinyl, carbonyl, carbamide, carbamate,
carbonate, ester (i.e., monoester, diester), or other linker as is
generally known in the art.
[0115] In one embodiment, a formulation of the invention (e.g., a
formulated molecular compositions (FMC) or lipid nanoparticle (LNP)
of the invention) is a neutral lipid having any of formulae
NLI-NLVII.
[0116] Examples of a steroid hormone include those comprising
cholesterol, estrogen, testosterone, progesterone, glucocortisone,
adrenaline, insulin, glucagon, cortisol, vitamin D, thyroid
hormone, retinoic acid, and/or growth hormones.
[0117] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, other nucleic acid
molecule or other biologically active molecule described herein), a
cationic lipid, a neutral lipid, and a polyethyleneglycol
conjugate, such as a PEG-diacylglycerol, PEG-diacylglycamide,
PEG-cholesterol, or PEG-DMB conjugate. In another embodiment, the
composition further comprises cholesterol or a cholesterol
derivative. The compositions described herein are generally
referred to as formulated molecular compositions (FMC) or lipid
nanoparticles (LNP). In some embodiments of the invention, a
formulated molecular composition (FMC) or lipid nanoparticle (LNP)
composition further comprises cholesterol or a cholesterol
derivative.
[0118] Suitable cationic lipid include those cationic lipids which
carry a net negative charge at a selected pH, such as physiological
pH. Particularly useful cationic lipids include those having a
relatively small head group, such as a tertiary amine, quaternary
amine or guanidine head group, and sterically hindered asymmetric
lipid chains. In any of the embodiments described herein, the
cationic lipid can be selected from those comprising Formulae CLI,
CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI,
CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVI, CLXVII, CLXVIII, CLXIX,
CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII,
CLXXVIII, CLXXIX, CLXXX, CLXXXI, CLXXXII, CLXXXIII, CLXXXIV,
CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI,
CLXXXXII CLXXXX, CLXXXXI, CLXXXXII;
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),
1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),
1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),
Dioleoyloxy-N-[2-sperminecarboxamido)ethyl}-N,N-dimethyl-1-propanaminiumt-
rifluoroacetate (DOSPA), Dioctadecylamidoglycyl spermine (DOGS),
DC-Chol, 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium
bromide (DMRIE),
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-dimethy-1-(cis,cis-9',1-
2'-octadecadienoxy)propane (CpLinDMA),
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA),
1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), and/or
a mixture thereof, as well as other cationic lipids sharing similar
properties. The above cationic lipids can include various differing
salts as are known in the art. Non-limiting examples of these
cationic lipid structures are shown in FIGS. 1-5 and FIG. 19.
[0119] In some embodiments, the head group of the cationic lipid
can be attached to the lipid chain via a cleavable or non-cleavable
linker, such as a linker described herein or otherwise known in the
art. Non-limiting examples of suitable linkers include those
comprising a C1 to C10 alkyl, alkyl ether, polyether, polyethylene
glycol, acetal, amide, carbonyl, carbamide, carbamate, carbonate,
ester (i.e., monoester, diester), or succinyl.
[0120] Suitable neutral lipids include those comprising any of a
variety of neutral uncharged, zwitterionic or anionic lipids
capable of producing a stable complex. They are preferably neutral,
although they can alternatively be positively or negatively
charged. In any of the embodiments described herein, suitable
neutral lipids include those selected from compounds having
formulae NLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), -phosphatidylet-hanolamine
(POPE) and dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
cholesterol, as well as other neutral lipids described herein
below, and/or a mixture thereof.
[0121] Suitable polyethyleneglycol-diacylglycerol or
polyethyleneglycol-diacylglycamide (PEG-DAG) conjugates include
those comprising a dialkylglycerol or dialkylglycamide group having
alkyl chain length independently comprising from about C4 to about
C40 saturated or unsaturated carbon atoms. The dialkylglycerol or
dialkylglycamide group can further comprise one or more substituted
alkyl groups. In any of the embodiments described herein, the PEG
conjugate can be selected from PEG-dilaurylglycerol (C12),
PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16),
PEG-disterylglycerol (C18), PEG-dilaurylglycamide (C12),
PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), and
PEG-disterylglycamide (C18), PEG-cholesterol
(1-[8'-(Cholest-5-en-3.beta.-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-
-.omega.-methyl-poly(ethylene glycol), and PEG-DMB
(3,4-Ditetradecoxylbenzyl-.omega.-methyl-poly(ethylene
glycol)ether).
[0122] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule) formulated as L051, L053, L054, L060, L061, L069,
L073, L077, L080, L082, L083, L086, L097, L098, L099, L100, L101,
L102, L103, L104, L105, L106, L107, L108, L109, L110, L111, L112,
L113, L114, L115, L116, L117, L118, L121, L122, L123, L124, L130,
L131, L132, L133, L134, L149, L155, L156, L162, L163, L164, L165,
L166, L167, L174, L175, L176, L180, L181, and/or L182 herein (see
Table IV).
[0123] Other suitable PEG conjugates include PEG-cholesterol or
PEG-DMB conjugates (see for example FIG. 24). In one embodiment,
PEG conjugates include PEGs attached to saturated or unsaturated
lipid chains such as oleyl, linoleyl and similar lipid chains.
[0124] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid having any of Formulae
CLI-CLXXXXII, a neutral lipid, and a PEG-DAG (i.e.,
polyethyleneglycol-diacylglycerol or
polyethyleneglycol-diacylglycamide), PEG-cholesterol, or PEG-DMB
conjugate. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative. In another embodiment, the
composition is formulated as L051, L053, L054, L060, L061, L069,
L073, L077, L080, L082, L083, L086, L097, L098, L099, L100, L101,
L102, L103, L104, L105, L106, L107, L108, L109, L110, L111, L112,
L113, L114, L115, L116, L117, L118, L121, L122, L123, L124, L130,
L131, L132, L133, L134, L149, L155, L156, L162, L163, L164, L165,
L166, L167, L174, L175, L176, L180, L181, and/or L182 herein (see
Table IV).
[0125] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising
3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA), a neutral lipid comprising
distearoylphosphatidylcholine (DSPC), a PEG-DAG comprising
PEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In one
embodiment, the molar ratio of CLinDMA:DSPC:cholesterol:PEG-DMG are
48:40:10:2 respectively, this composition is generally referred to
herein as formulation L051.
[0126] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), a neutral lipid
comprising distearoylphosphatidylcholine (DSPC), a PEG-DAG
comprising PEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In
one embodiment, the molar ratio of DMOBA:DSPC:cholesterol:PEG-DMG
are 30:20:48:2 respectively, this composition is generally referred
to herein as formulation L053.
[0127] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), a neutral lipid
comprising distearoylphosphatidylcholine (DSPC), a PEG-DAG
comprising PEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In
one embodiment, the molar ratio of DMOBA:DSPC:cholesterol:PEG-DMG
are 50:20:28:2 respectively, this composition is generally referred
to herein as formulation L054. In another embodiment, the
composition further comprises a neutral lipid, such as
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof.
[0128] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising comprising
3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA), a cationic lipid comprising
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), a neutral lipid
comprising distearoylphosphatidylcholine (DSPC), a PEG-DAG
comprising PEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In
one embodiment, the molar ratio of
CLinDMA:DMOBA:DSPC:cholesterol:PEG-DMG are 25:25:20:28:2
respectively, this composition is generally referred to herein as
formulation L073.
[0129] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising comprising
3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA), a neutral lipid comprising
distearoylphosphatidylcholine (DSPC), a PEG comprising
PEG-Cholesterol (PEG-Chol), and cholesterol. In one embodiment, the
molar ratio of CLinDMA:DSPC:cholesterol:PEG-Chol are 48:40:10:2
respectively, this composition is generally referred to herein as
formulation L069.
[0130] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising comprising
1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), a
neutral lipid comprising distearoylphosphatidylcholine (DSPC), a
PEG-DAG comprising PEG-n-dimyristylglycerol (PEG-DMG), and
cholesterol. In one embodiment, the molar ratio of
DOcarbDAP:DSPC:cholesterol:PEG-DMG are 30:20:48:2 respectively,
this composition is generally referred to herein as formulation
T018.1.
[0131] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), a cationic lipid comprising comprising
N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), a neutral lipid
comprising distearoylphosphatidylcholine (DSPC), a PEG-DAG
comprising PEG-n-dimyristylglycerol (PEG-DMG), and cholesterol. In
one embodiment, the molar ratio of DODMA:DSPC:cholesterol:PEG-DMG
are 30:20:48:2 respectively, this composition is generally referred
to herein as formulation T019.1.
[0132] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), and a cationic lipid comprising a compound having
any of Formula CLI, CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII,
CLIX, CLX, CLXI, CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVII,
CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV,
CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI, CLXXXII,
CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII, CLXXXIX,
CLXXXX, CLXXXXI, CLXXXXII CLXXXX, CLXXXXI, or CLXXXXII;. In another
embodiment, the composition further comprises a neutral lipid, such
as dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. In another embodiment, the composition further
comprises a PEG conjugate. In yet another embodiment, the
composition further comprises cholesterol or a cholesterol
derivative.
[0133] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), and a cationic lipid comprising
3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA). In another embodiment, the
composition further comprises a neutral lipid, such as
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. In another embodiment, the composition further
comprises a PEG conjugate (i.e., polyethyleneglycol diacylglycerol
(PEG-DAG), PEG-cholesterol, or PEG-DMB). In yet another embodiment,
the composition further comprises cholesterol or a cholesterol
derivative.
[0134] In one embodiment, the invention features a composition
comprising a biologically active molecule (e.g., a polynucleotide
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule), and a cationic lipid comprising
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA). In another
embodiment, the composition further comprises a neutral lipid, such
as dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. In yet another embodiment, the composition further
comprises the cationic lipid CLinDMA. In another embodiment, the
composition further comprises a PEG conjugate. In yet another
embodiment, the composition further comprises cholesterol or a
cholesterol derivative.
[0135] The term "biologically active molecule" as used herein
refers to compounds or molecules that are capable of eliciting or
modifying a biological response in a system. Non-limiting examples
of biologically active molecules include antibodies (e.g.,
monoclonal, chimeric, humanized etc.), cholesterol, hormones,
antivirals, peptides, proteins, chemotherapeutics, small molecules,
vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,
enzymatic nucleic acids, antisense nucleic acids, triplex forming
oligonucleotides, 2,5-A chimeras, siNA, siRNA, miRNA, RNAi
inhibitors, dsRNA, allozymes, aptamers, decoys and analogs thereof.
Biologically active molecules of the invention also include
molecules capable of modulating the pharmacokinetics and/or
pharmacodynamics of other biologically active molecules, for
example, lipids and polymers such as polyamines, polyamides,
polyethylene glycol and other polyethers. In certain embodiments,
the term biologically active molecule is used interchangeably with
the term "molecule" or "molecule of interest" herein.
[0136] In one embodiment, the invention features a composition
comprising a siNA molecule, a cationic lipid having any of Formulae
CLI-CLXXXXII, a neutral lipid, and a
polyethyleneglycol-diacylglycerol or
polyethyleneglycol-diacylglycamide (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB). These compositions are generally referred to herein as
formulated siNA compositions. In another embodiment, a formulated
siNA composition of the invention further comprises cholesterol or
a cholesterol derivative.
[0137] In one embodiment, the siNA component of a formulated siNA
composition of the invention is chemically modified so as not to
stimulate an interferon response in a mammalian cell, subject, or
organism. Such siNA molecules can be said to have improved
toxicologic profiles, such as having attenuated or no
immunostimulatory properties, having attenuated or no off-target
effect, or otherwise as described herein.
[0138] In one embodiment, the invention features a composition
comprising a miRNA molecule, a cationic lipid having any of
Formulae CLI-CLXXXXII, a neutral lipid, and a
polyethyleneglycol-diacylglycerol or
polyethyleneglycol-diacylglycamide (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB). These compositions are generally referred to herein as
formulated miRNA compositions. In another embodiment, a formulated
miRNA composition of the invention further comprises cholesterol or
a cholesterol derivative.
[0139] In one embodiment, the miRNA component of a formulated miRNA
composition of the invention is chemically modified so as not to
stimulate an interferon response in a mammalian cell, subject, or
organism. Such miRNA molecules can be said to have improved
toxicologic profiles, such as having attenuated or no
immunostimulatory properties, having attenuated or no off-target
effect, or otherwise as described herein.
[0140] In one embodiment, the invention features a composition
comprising a RNAi inhibitor molecule, a cationic lipid having any
of Formulae CLI-CLXXXXII, a neutral lipid, and a
polyethyleneglycol-diacylglycerol or
polyethyleneglycol-diacylglycamide (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB). These compositions are generally referred to herein as
formulated RNAi inhibitor compositions. In another embodiment, a
formulated RNAi inhibitor composition of the invention further
comprises cholesterol or a cholesterol derivative.
[0141] In one embodiment, the RNAi inhibitor component of a
formulated RNAi inhibitor composition of the invention is
chemically modified so as not to stimulate an interferon response
in a mammalian cell, subject, or organism. Such RNAi inhibitor
molecules can be said to have improved toxicologic profiles, such
as having attenuated or no immunostimulatory properties, having
attenuated or no off-target effect, or otherwise as described
herein
[0142] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a
polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB); and (d) a short interfering nucleic acid (siNA) molecule
that mediates RNA interference (RNAi) against RNA of a target gene,
wherein each strand of said siNA molecule is about 18 to about 28
nucleotides in length; and one strand of said siNA molecule
comprises nucleotide sequence having sufficient complementarity to
the target gene RNA for the siNA molecule to mediate RNA
interference against the target gene RNA. In one embodiment, the
target RNA comprises RNA sequence referred to by Genbank Accession
numbers in International PCT Publication No. WO 03/74654, serial
No. PCT/US03/05028, and U.S. patent appliation Ser. No. 10/923,536
both incorporated by reference herein. In another embodiment, the
composition further comprises cholesterol or a cholesterol
derivative.
[0143] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a
polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB); and (d) a miRNA molecule that mediates RNA interference
(RNAi) against RNA of a target gene, wherein each strand of said
miRNA molecule is about 18 to about 40 nucleotides in length; and
one strand of said miRNA molecule comprises nucleotide sequence
having sufficient complementarity to the target gene RNA for the
miRNA molecule to mediate RNA interference against the target gene
RNA. In one embodiment, the target RNA comprises RNA sequence
referred to by Genbank Accession numbers in International PCT
Publication No. WO 03/74654, serial No. PCT/US03/05028, and U.S.
patent appliation Ser. No. 10/923,536 both incorporated by
reference herein. In another embodiment, the composition further
comprises cholesterol or a cholesterol derivative.
[0144] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a
polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB); and (d) a RNAi inhibitor molecule that modulates RNA
interference (RNAi) activity of a miRNA or siRNA target, wherein
said RNAi inhibitor molecule is about 15 to about 40 nucleotides in
length; and said RNAi inhibitor molecule comprises nucleotide
sequence having sufficient complementarity to the target siRNA or
miRNA for the RNAi inhibitor molecule to modulate the RNAi activity
of the target siRNA or miRNA. In one embodiment, the miRNA or siRNA
target comprises RNA sequence comprising a portion of RNA sequence
referred to by Genbank Accession numbers in International PCT
Publication No. WO 03/74654, serial No. PCT/US03/05028, and U.S.
patent appliation Ser. No. 10/923,536 both incorporated by
reference herein. In another embodiment, the composition further
comprises cholesterol or a cholesterol derivative.
[0145] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a
polyethyleneglycol-diacylglycerol (PEG-DAG) conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB); and (d) a short interfering nucleic acid (siNA) molecule
that mediates RNA interference (RNAi) against a Hepatitis Virus
RNA, wherein each strand of said siNA molecule is about 18 to about
28 nucleotides in length; and one strand of said siNA molecule
comprises nucleotide sequence having sufficient complementarity to
the Hepatitis Virus RNA for the siNA molecule to mediate RNA
interference against the Hepatitis Virus RNA. In one embodiment,
the Hepatitis Virus RNA is Hepatitis B Virus (HBV). In one
embodiment, the Hepatitis Virus RNA is Hepatitis C Virus (HCV). In
one embodiment, the siNA comprises sequences described in U.S.
patent application Ser. Nos. 60/401104, 10/667,271, and 10/942,560,
which are incorporated by reference in their entireties herein. In
another embodiment, the composition further comprises cholesterol
or a cholesterol derivative.
[0146] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
Protein Tyrosine Phosphatase 1B (PTP1B) RNA, wherein each strand of
said siNA molecule is about 18 to about 28 nucleotides in length;
and one strand of said siNA molecule comprises nucleotide sequence
having sufficient complementarity to the PTP1B RNA for the siNA
molecule to mediate RNA interference against the PTP1B RNA. In one
embodiment, the siNA comprises sequences described in U.S. Patent
Application Publication Nos. 20040019001 and 200500704978, which
are incorporated by reference in their entireties herein. In
another embodiment, the composition further comprises cholesterol
or a cholesterol derivative.
[0147] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
Transforming Growth Factor beta (TGF-beta) and/or Transforming
Growth Factor beta Receptor (TGF-betaR) RNA, wherein each strand of
said siNA molecule is about 18 to about 28 nucleotides in length;
and one strand of said siNA molecule comprises nucleotide sequence
having sufficient complementarity to the TGF-beta and/or TGF-betaR
RNA for the siNA molecule to mediate RNA interference against the
TGF-beta and/or TGF-betaR RNA. In one embodiment, the siNA
comprises sequences described in U.S. Ser. No. 11/054,047, which is
incorporated by reference in their entireties herein. In another
embodiment, the composition further comprises cholesterol or a
cholesterol derivative.
[0148] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
cholesteryl ester transfer protein (CETP) RNA, wherein each strand
of said siNA molecule is about 18 to about 28 nucleotides in
length; and one strand of said siNA molecule comprises nucleotide
sequence having sufficient complementarity to the CETP RNA for the
siNA molecule to mediate RNA interference against the CETP RNA. In
one embodiment, the siNA comprises sequences described in U.S. Ser.
No. 10/921,554, which is incorporated by reference in its entirety
herein. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative.
[0149] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
Gastric Inhibitory Peptide (GIP) RNA, wherein each strand of said
siNA molecule is about 18 to about 28 nucleotides in length; and
one strand of said siNA molecule comprises nucleotide sequence
having sufficient complementarity to the GIP RNA for the siNA
molecule to mediate RNA interference against the GIP RNA. In one
embodiment, the siNA comprises sequences described in U.S. Ser. No.
10/916,030, which is incorporated by reference in its entirety
herein. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative.
[0150] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
Stearoyl-CoA Desaturase (SCD) RNA, wherein each strand of said siNA
molecule is about 18 to about 28 nucleotides in length; and one
strand of said siNA molecule comprises nucleotide sequence having
sufficient complementarity to the SCD RNA for the siNA molecule to
mediate RNA interference against the SCD RNA. In one embodiment,
the siNA comprises sequences described in U.S. Ser. No. 10/923,451,
which is incorporated by reference in its entirety herein. In
another embodiment, the composition further comprises cholesterol
or a cholesterol derivative.
[0151] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a
polyethyleneglycol-diacylglycerol conjugate (i.e.,
polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or
PEG-DMB); and (d) a short interfering nucleic acid (siNA) molecule
that mediates RNA interference (RNAi) against Acetyl-CoA
carboxylase (ACACB) RNA, wherein each strand of said siNA molecule
is about 18 to about 28 nucleotides in length; and one strand of
said siNA molecule comprises nucleotide sequence having sufficient
complementarity to the ACACB RNA for the siNA molecule to mediate
RNA interference against the ACACB RNA. In one embodiment, the siNA
comprises sequences described in U.S. Ser. No. 10/888,226, which is
incorporated by reference in its entirety herein. In another
embodiment, the composition further comprises cholesterol or a
cholesterol derivative.
[0152] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
apolipoprotein RNA (e.g., apo AI, apo A-IV, apo B, apo C-III,
and/or apo E RNA), wherein each strand of said siNA molecule is
about 18 to about 28 nucleotides in length; and one strand of said
siNA molecule comprises nucleotide sequence having sufficient
complementarity to the apolipoprotein RNA for the siNA molecule to
mediate RNA interference against the apolipoprotein RNA. In one
embodiment, the siNA comprises sequences described in U.S. Ser. No.
11/054,047, which is incorporated by reference in their entireties
herein. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative.
[0153] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
VEGF and/or VEGF-receptor RNA (e.g., VEGF, VEGFR1, VEGFR2 and/or
VEGFR3 RNA), wherein each strand of said siNA molecule is about 18
to about 28 nucleotides in length; and one strand of said siNA
molecule comprises nucleotide sequence having sufficient
complementarity to the VEGF and/or VEGF-receptor RNA for the siNA
molecule to mediate RNA interference against the VEGF and/or
VEGF-receptor RNA. In one embodiment, the siNA comprises sequences
described in U.S. Ser. No. 10/962,898, which is incorporated by
reference in their entireties herein. In another embodiment, the
composition further comprises cholesterol or a cholesterol
derivative.
[0154] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG_DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
IL4-receptor RNA, wherein each strand of said siNA molecule is
about 18 to about 28 nucleotides in length; and one strand of said
siNA molecule comprises nucleotide sequence having sufficient
complementarity to the IL4-receptor RNA for the siNA molecule to
mediate RNA interference against the IL4-receptor RNA. In one
embodiment, the siNA comprises sequences described in U.S. Ser. No.
11/001,347, which is incorporated by reference in their entireties
herein. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative.
[0155] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG_DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
Hairless RNA, wherein each strand of said siNA molecule is about 18
to about 28 nucleotides in length; and one strand of said siNA
molecule comprises nucleotide sequence having sufficient
complementarity to the Hairless RNA for the siNA molecule to
mediate RNA interference against the Hairless RNA. In one
embodiment, the siNA comprises sequences described in U.S. Ser. No.
10/919,964, which is incorporated by reference in their entireties
herein. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative.
[0156] In one embodiment, the invention features a composition
comprising: (a) a cationic lipid having any of Formulae
CLI-CLXXXXII; (b) a neutral lipid; (c) a polyethyleneglycol
conjugate (i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG_DMB); and (d) a short interfering nucleic
acid (siNA) molecule that mediates RNA interference (RNAi) against
a target RNA, wherein each strand of said siNA molecule is about 18
to about 28 nucleotides in length; and one strand of said siNA
molecule comprises nucleotide sequence having sufficient
complementarity to the target RNA for the siNA molecule to mediate
RNA interference against the target RNA. In one embodiment, the
target RNA comprises RNA sequence referred to by Genbank Accession
numbers in International PCT Publication No. WO 03/74654, serial
No. PCT/US03/05028, and U.S. patent appliation Ser. No. 10/923,536
both incorporated by reference herein. In another embodiment, the
composition further comprises cholesterol or a cholesterol
derivative.
[0157] In one embodiment, the cationic lipid component (e.g., a
compound having any of Formulae CLI-CLXXXXII or as otherwise
described herein) of a composition of invention comprises from
about 2% to about 60%, from about 5% to about 45%, from about 5% to
about 15%, or from about 40% to about 50% of the total lipid
present in the formulation.
[0158] In one embodiment, the neutral lipid component of a
composition of the invention comprises from about 5% to about 90%,
or from about 20% to about 85% of the total lipid present in the
formulation.
[0159] In one embodiment, the PEG conjugate (i.e., PEG_DAG,
PEG-cholesterol, PEG-DMB) of a composition of the invention
comprises from about 1% to about 20%, or from about 4% to about 15%
of the total lipid present in the formulation.
[0160] In one embodiment, the cholesterol component of a
composition of the invention comprises from about 10% to about 60%,
or from about 20% to about 45% of the total lipid present in the
formulation.
[0161] In one embodiment, a formulated siNA composition of the
invention comprises a cationic lipid component comprising from
about 30 to about 50% of the total lipid present in the
formulation, a neutral lipid comprising from about 30 to about 50%
of the total lipid present in the formulation, and a PEG conjugate
(i.e., PEG_DAG, PEG-cholesterol, PEG-DMB) comprising about 0 to
about 10% of the total lipid present in the formulation.
[0162] In one embodiment, a formulated molecular composition of the
invention comprises a biologically active molecule (e.g., a
polynucleotide such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule), a compound having any of Formulae
CLI-CLXXXXII, DSPC, and a PEG conjugate (i.e., PEG-DAG,
PEG-cholesterol, PEG-DMB). In one embodiment, the PEG conjugate is
PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),
PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In
another embodiment, the PEG conjugate is PEG-dilaurylglycamide
(C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide
(C16), or PEG-disterylglycamide (C18). In another embodiment, the
PEG conjugate is PEG-cholesterol or PEG-DMB. In another embodiment,
the formulated molecular composition further comprises cholesterol
or a cholesterol derivative.
[0163] In one embodiment, a formulated molecular composition of the
invention comprises a biologically active molecule (e.g., a
polynucleotide such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule), a compound having Formula CLI, DSPC,
and a PEG conjugate. In one embodiment, the PEG conjugate is
PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),
PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In
another embodiment, the PEG conjugate is PEG-dilaurylglycamide
(C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide
(C16), or PEG-disterylglycamide (C18). In another embodiment, the
PEG conjugate is PEG-cholesterol or PEG-DMB. In another embodiment,
the formulated molecular composition further comprises cholesterol
or a cholesterol derivative.
[0164] In one embodiment, a formulated molecular composition of the
invention comprises a biologically active molecule (e.g., a
polynucleotide such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule), a compound having Formula CLV, DSPC,
and a PEG conjugate. In one embodiment, the PEG conjugate is
PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),
PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In
another embodiment, the PEG conjugate is PEG-dilaurylglycamide
(C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide
(C16), or PEG-disterylglycamide (C18). In another embodiment, the
PEG conjugate is PEG-cholesterol or PEG-DMB. In another embodiment,
the formulated molecular composition further comprises cholesterol
or a cholesterol derivative.
[0165] In one embodiment, a composition of the invention (e.g., a
formulated molecular composition) further comprises a targeting
ligand for a specific cell of tissue type. Non-limiting examples of
such ligands include sugars and carbohydrates such as galactose,
galactosamine, and N-acetyl galactosamine; hormones such as
estrogen, testosterone, progesterone, glucocortisone, adrenaline,
insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic
acid, and growth hormones; growth factors such as VEGF, EGF, NGF,
and PDGF; cholesterol; bile acids; neurotransmitters such as GABA,
Glutamate, acetylcholine; NOGO; inostitol triphosphate;
diacylglycerol; epinephrine; norepinephrine; Nitric Oxide,
peptides, vitamins such as folate and pyridoxine, drugs, antibodies
and any other molecule that can interact with a receptor in vivo or
in vitro. The ligand can be attached to any component of a
formulated siNA composition of invention (e.g., cationic lipid
component, neutral lipid component, PEG-DAG component, or siNA
component etc.) using a linker molecule, such as an amide, amido,
carbonyl, ester, peptide, disulphide, silane, nucleoside, abasic
nucleoside, polyether, polyamine, polyamide, peptide, carbohydrate,
lipid, polyhydrocarbon, phosphate ester, phosphoramidate,
thiophosphate, alkylphosphate, or photolabile linker. In one
embodiment, the linker is a biodegradable linker.
[0166] In one embodiment, the PEG conjugate of the invention, such
as a PEG-DAG, PEG-cholesterol, PEG-DMB , comprises a 200 to 10,000
atom PEG molecule.
[0167] In one embodiment, the compositions of the present
invention, e.g., a formulated molecular composition, comprise a
diacylglycerol-polyethyleneglycol conjugate, i.e., a DAG-PEG
conjugate. The term "diacylglycerol" refers to a compound having
2-fatty acyl chains, R1 and R2, 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. Diacylglycerols have the
following general Formula VIII:
##STR00051##
[0168] wherein R1 and R2 are each an alkyl, substituted alkyl,
aryl, substituted aryl, lipid, or a ligand. In one embodiment, R1
and R2 are each independently a C2 to C30 alkyl group. In one
embodiment, the DAG-PEG conjugate is a dilaurylglycerol (C12)-PEG
conjugate, a dimyristylglycerol (C14)-PEG conjugate, a
dipalmitoylglycerol (C16)-PEG conjugate, a disterylglycerol
(C18)-PEG conjugate, PEG-dilaurylglycamide (C12),
PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), or
PEG-disterylglycamide (C18). Those of skill in the art will readily
appreciate that other diacylglycerols can be used in the DAG-PEG
conjugates of the present invention.
[0169] In one embodiment, the compositions of the present
invention, e.g., a formulated molecular composition, comprise a
polyethyleneglycol-cholesterol conjugate, i.e., a PEG-chol
conjugate. The PEG-chol conjugate can comprise a 200 to 10,000 atom
PEG molecule linked to cholesterol or a cholesterol derivative. An
exemplary PEG-chol and the synthesis thereof is shown in FIG.
24.
[0170] In one embodiment, the compositions of the present
invention, e.g., a formulated molecular composition, comprise a
polyethyleneglycol-DMB conjugate. The term "DMB" refers to the
compound 3,4-Ditetradecoxylbenzyl-.beta.-methyl-poly(ethylene
glycol) ether. The PEG-DMB conjugate can comprise a 200 to 10,000
atom PEG molecule linked to DMB. An exemplary PEG-DMB and the
synthesis thereof is shown in FIG. 24A.
[0171] In one embodiment, the compositions of the present
invention, e.g., a formulated molecular composition, comprise a
polyethyleneglycol-DMG (PEG-DMG) conjugate. The term "PEG-DMG" can
refer to the compound
1-[8'-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbam-
oyl-.omega.-methyl-poly(ethylene glycol). The PEG-DMG conjugate can
comprise a 200 to 10,000 atom PEG molecule linked to DMG moiety. In
one embodiment, PEG is a polydispersion represented by the formula
PEG.sub.n, where n=about 33 to 67 for a 1500 Da to 3000 Da PEG,
average=45 for 2KPEG/PEG2000. An exemplary PEG-DMG and the
synthesis thereof is shown in FIG. 24B.
[0172] The term "ligand" refers to any compound or molecule, such
as a drug, peptide, hormone, or neurotransmitter that is capable of
interacting with another compound, such as a receptor, either
directly or indirectly. The receptor that interacts with a ligand
can be present on the surface of a cell or can alternately be an
intercellular receptor. Interaction of the ligand with the receptor
can result in a biochemical reaction, or can simply be a physical
interaction or association. Non-limiting examples of ligands
include sugars and carbohydrates such as galactose, galactosamine,
and N-acetyl galactosamine; hormones such as estrogen,
testosterone, progesterone, glucocortisone, adrenaline, insulin,
glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid, and
growth hormones; growth factors such as VEGF, EGF, NGF, and PDGF;
cholesterol; bile acids; neurotransmitters such as GABA, Glutamate,
acetylcholine; NOGO; inostitol triphosphate; diacylglycerol;
epinephrine; norepinephrine; Nitric Oxide, peptides, vitamins such
as folate and pyridoxine, drugs, antibodies and any other molecule
that can interact with a receptor in vivo or in vitro. The ligand
can be attached to a compound of the invention using a linker
molecule, such as an amide, amido, carbonyl, ester, peptide,
disulphide, silane, nucleoside, abasic nucleoside, polyether,
polyamine, polyamide, peptide, carbohydrate, lipid,
polyhydrocarbon, phosphate ester, phosphoramidate, thiophosphate,
alkylphosphate, or photolabile linker. In one embodiment, the
linker is a biodegradable linker.
[0173] The term "degradable linker" as used herein, refers to
linker moieties that are capable of cleavage under various
conditions. Conditions suitable for cleavage can include but are
not limited to pH, UV irradiation, enzymatic activity, temperature,
hydrolysis, elimination, and substitution reactions, and
thermodynamic properties of the linkage.
[0174] The term "photolabile linker" as used herein, refers to
linker moieties as are known in the art that are selectively
cleaved under particular UV wavelengths. Compounds of the invention
containing photolabile linkers can be used to deliver compounds to
a target cell or tissue of interest, and can be subsequently
released in the presence of a UV source.
[0175] The term "lipid" as used herein, refers to any lipophilic
compound. Non-limiting examples of lipid compounds include fatty
acids and their derivatives, including straight chain, branched
chain, saturated and unsaturated fatty acids, carotenoids,
terpenes, bile acids, and steroids, including cholesterol and
derivatives or analogs thereof.
[0176] In addition to the foregoing components, the compositions of
the present invention can further comprise cationic poly(ethylene
glycol) (PEG) lipids, or CPLs, that have been designed for
insertion into lipid bilayers to impart a positive charge (see for
example Chen, et al., 2000, Bioconj. Chem. 11, 433-437). Suitable
formulations for use in the present invention, and methods of
making and using such formulations are disclosed, for example in
U.S. application Ser. No. 09/553,639, which was filed Apr. 20,
2000, and PCT Patent Application No. CA 00/00451, which was filed
Apr. 20, 2000 and which published as WO 00/62813 on Oct. 26, 2000,
the teachings of each of which is incorporated herein in its
entirety by reference.
[0177] In one embodiment, the compositions of the present
invention, i.e., those formulated molecular compositions containing
PEG conjugates, are made using any of a number of different
methods. In one embodiment, the present invention provides
lipid-nucleic acid particles produced via hydrophobic
polynucleotide-lipid intermediate complexes. The complexes are
preferably charge-neutralized. Manipulation of these complexes in
either detergent-based or organic solvent-based systems can lead to
particle formation in which the nucleic acid is protected.
[0178] In one embodiment, the present invention provides a
serum-stable formulated molecular composition (e.g., comprising a
biologically active molecules such as polynucleotides including
siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme,
2-5A, triplex forming oligonucleotide, or other nucleic acid
molecules) in which the biologically active molecule is
encapsulated in a lipid bilayer and is protected from degradation
(for example, where the composition adopts a lamellar structure).
Additionally, the formulated particles formed in the present
invention are preferably neutral or negatively-charged at
physiological pH. In one embodiment, for in vivo applications,
neutral particles can be advantageous, while for in vitro
applications the particles can be negatively charged. This provides
the further advantage of reduced aggregation over the
positively-charged liposome formulations in which a biologically
active molecule can be encapsulated in cationic lipids.
[0179] In addition, the present invention provides serum-stable
formulated molecular compositions that undergo a rapid pH-dependent
phase transition. The pH-dependent phase transition results in a
structural change that increases the efficiency of delivery of a
biologically active molecule, such as a polynucleotide, into a
biological system, such as a cell. The structural change can
increase the efficiency of delivery by, for example, increasing
cell membrane fusion and release of a biologically active molecule
into a biological system. Thus, in one embodiment, the serum-stable
formulated molecular composition is stable in plasma or serum
(i.e., in circulation) and stable at physiologic pH (i.e., about pH
7.4) and undergoes a rapid pH-dependent phase transition resulting
in a structural change that increases the efficiency of delivery of
a biologically active molecule into a biological system. In one
embodiment, the pH dependent phase transition occurs at about pH
5.5-6.5. In one embodiment, the serum-stable formulated molecular
composition undergoes a structural change to adopt an inverted
hexagonal structure at about pH 5.5-6.5. For example, the
serum-stable formulated molecular composition can transition from a
stable lamellar structure adopted in circulation (i.e., in plasma
or serum) at physiologic pH (about pH 7.4) to a less stable and
more efficient delivery composition having an inverted hexagonal
structure at pH 5.5-6.5, which is the pH found in the early
endosome. The serum-stable formulated molecular compositions that
undergo a rapid pH-dependent phase transition demonstrate increased
efficiency in the delivery of biologically active molecules due to
their stability in circulation at physiologic pH and their ability
to undergo a pH dependent structural change that increases cell
membrane fusion and release of a biologically active molecule into
a biological system, such as a cell.
[0180] The serum-stable formulated molecular composition that
undergoes a rapid pH-dependent phase transition comprises a
biologically active molecule (e.g., a polynucleotide such as a
siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme,
2-5A, triplex forming oligonucleotide, other nucleic acid molecule
or other biologically active molecule described herein), a cationic
lipid, a neutral lipid, and a polyethylene conjugate such as a
polyethyleneglycol-diacylglycerol,
polyethyleneglycol-diacylglycamide, polyethyleneglycol-cholesterol
or polyethylene-DMB conjugate. In another embodiment, the
composition further comprises cholesterol or a cholesterol
derivative. Examples of suitable cationic lipids, neutral lipids,
and PEG conjugates are provided herein.
[0181] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is CLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
PEG-DMG. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative. This is known as
formulation L051 (see Table IV).
[0182] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
PEG-DMG. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative. This is known as
formulation L053 or L054 (see Table IV).
[0183] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is CLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-cholesterol. In another embodiment, the composition further
comprises cholesterol or a cholesterol derivative. This is known as
formulation L069 (see Table IV).
[0184] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is pCLinDMA or CLinDMA and DMOBA, the neutral lipid
is distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
PEG-DMG. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative. This is known as
formulation L073 (see Table N).
[0185] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is eCLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-cholesterol. In another embodiment, the composition further
comprises cholesterol or a cholesterol derivative. This is known as
formulation L077 (see Table IV).
[0186] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is eCLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-DMG. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative. This is known as
formulation L080 (see Table IV).
[0187] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is pCLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-DMG. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative. This is known as
formulation L082 (see Table IV).
[0188] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is pCLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-cholesterol. In another embodiment, the composition further
comprises cholesterol or a cholesterol derivative. This is known as
formulation L083 (see Table IV).
[0189] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is CLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-DMG. In another embodiment, the composition further comprises
cholesterol or a cholesterol derivative and Linoleyl alcohol. This
is known as formulation L086 (see Table IV).
[0190] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMLBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2KPEG-DMG. This is known as formulation L061 (see
Table IV).
[0191] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2KPEG-DMG, and the nitrogen to phoshpate (N/P)
ratio of the formulated molecular composition is 5. This is known
as formulation L060 (see Table IV).
[0192] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMLBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2KPEG-DMG. This is known as formulation L097 (see
Table IV).
[0193] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2KPEG-DMG, and the nitrogen to phoshpate (N/P)
ratio of the formulated molecular composition is 3. This is known
as formulation L098 (see Table IV).
[0194] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2KPEG-DMG, and the nitrogen to phoshpate (N/P)
ratio of the formulated molecular composition is 4. This is known
as formulation L099 (see Table IV).
[0195] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is DOBA, and the PEG
conjugate is 2KPEG-DMG (3%), and the nitrogen to phoshpate (N/P)
ratio of the formulated molecular composition is 3. This is known
as formulation L100 (see Table IV).
[0196] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2K-PEG-Cholesterol. This is known as formulation
L101 (see Table IV).
[0197] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMOBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2K-PEG-Cholesterol, and the nitrogen to phoshpate
(N/P) ratio of the formulated molecular composition is 5. This is
known as formulation L102 (see Table IV).
[0198] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is DMLBA, the neutral lipid is cholesterol, and the
PEG conjugate is 2K-PEG-Cholesterol. This is known as formulation
L103 (see Table IV).
[0199] In one embodiment, the invention features a serum-stable
formulated molecular composition comprising a biologically active
molecule (e.g., a siNA, miRNA, or RNAi inhibitor molecule), a
cationic lipid, a neutral lipid, and a PEG-conjugate, in which the
cationic lipid is CLinDMA, the neutral lipid is
distearoylphosphatidylcholine (DSPC), and the PEG conjugate is
2KPEG-cholesterol. In another embodiment, the composition further
comprises cholesterol or a cholesterol derivative and Linoleyl
alcohol. This is known as formulation L104 (see Table IV).
[0200] The invention additionally provides methods for determining
whether a formulated molecular composition will be effective for
delivery of a biologically active molecule into a biological
system. In one embodiment, the method for determining whether a
formulated molecular composition will be effective for delivery of
a biologically active molecule into a biological system comprises
(1) measuring the serum stability of the formulated molecular
composition and (2) measuring the pH dependent phase transition of
the formulated molecular composition, wherein a determination that
the formulated molecular composition is stable in serum and a
determination that the formulated molecular composition undergoes a
phase transition at about pH 4 to about 7, e.g., from 5.5 to 6.5,
indicates that the formulated molecular composition will be
effective for delivery of a biologically active molecule into a
biological system. In another embodiment, the method further
comprises measuring the transfection efficiency of the formulated
molecular composition in a cell in vitro.
[0201] The serum stability of the formulated molecular composition
can be measured using any assay that measures the stability of the
formulated molecular composition in serum, including the assays
described herein and otherwise known in the art. One exemplary
assay that can be used to measure the serum stability is an assay
that measures the relative turbidity of the composition in serum
over time. For example, the relative turbidity of a formulated
molecular composition can be determined by measuring the absorbance
of the formulated molecular composition in the presence or absence
of serum (i.e., 50%) at several time points over a 24 hour period
using a spectrophotometer. The formulated molecular composition is
stable in serum if the relative turbidity, as measured by
absorbance, remains constant at around 1.0 over time.
[0202] The pH dependent phase transition of the formulated
molecular composition can be measured using any assay that measures
the phase transition of the formulated molecular composition at
about pH 5.5-6.5, including the assays described herein and
otherwise known in the art. One exemplary assay that can be used to
measure the pH dependent phase transition is an assay that measures
the relative turbidity of the composition at different pH over
time. For example, the relative turbidity of a formulated molecular
composition can be determined by measuring the absorbance over time
of the formulated molecular composition in buffer having a range of
different pH values. The formulated molecular composition undergoes
pH dependent phase transition if the relative turbidity, as
measured by absorbance, decreases when the pH drops below 7.0.
[0203] In addition, the efficiency of the serum-stable formulated
molecular composition that undergoes a rapid pH-dependent phase
transition as a delivery agent can be determined by measuring the
transfection efficiency of the formulated molecular composition.
Methods for performing transfection assays are described herein and
otherwise known in the art.
[0204] In one embodiment, the particles made by the methods of this
invention have a size of about 50 to about 600 nm. The particles
can be formed by either a detergent dialysis method or by a
modification of a reverse-phase method which utilizes organic
solvents to provide a single phase during mixing of the components.
Without intending to be bound by any particular mechanism of
formation, a molecule (e.g., a biologically active molecule such as
a polynucleotide) is contacted with a detergent solution of
cationic lipids to form a coated molecular complex. These coated
molecules can aggregate and precipitate. However, the presence of a
detergent reduces this aggregation and allows the coated molecules
to react with excess lipids (typically, noncationic lipids) to form
particles in which the molecule of interest is encapsulated in a
lipid bilayer. The methods described below for the formation of
formulated molecular compositions using organic solvents follow a
similar scheme.
[0205] In one embodiment, the particles are formed using detergent
dialysis. Thus, the present invention provides a method for the
preparation of serum-stable formulated molecular compositions,
including those that undergo pH dependent phase transition,
comprising: (a) combining a molecule (e.g., a biologically active
molecule such as a polynucleotide, including siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecules) with
cationic lipids in a detergent solution to form a coated
molecule-lipid complex; (b) contacting noncationic lipids with the
coated molecule-lipid complex to form a detergent solution
comprising a siNA-lipid complex and noncationic lipids; and (c)
dialyzing the detergent solution of step (b) to provide a solution
of serum-stable molecule-lipid particles, wherein the molecule is
encapsulated in a lipid bilayer and the particles are serum-stable
and have a size of from about 50 to about 600 nm.
[0206] In one embodiment, an initial solution of coated
molecule-lipid (e.g., polynucleotide-lipid) complexes is formed,
for example, by combining the molecule with the cationic lipids in
a detergent solution.
[0207] In these embodiments, the detergent solution is preferably
an aqueous solution of a neutral detergent having a critical
micelle concentration of 15-300 mM, more preferably 20-50 mM.
Examples of suitable detergents include, for example,
N,N'-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide)
(BIGCHAP); BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol)
ether; Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8;
Mega 9; Zwittergent.RTM. 3-08; Zwittergent.RTM. 3-10; Triton X-405;
hexyl-, heptyl-, octyl- and nonyl-beta-D-glucopyranoside; and
heptylthioglucopyranoside; with octyl .beta.-D-glucopyranoside and
Tween-20 being the most preferred. The concentration of detergent
in the detergent solution is typically about 100 mM to about 2 M,
preferably from about 200 mM to about 1.5 M.
[0208] In one embodiment, the cationic lipids and the molecule of
interest (e.g., a biologically active molecule such as a
polynucleotide, including siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecules) will typically be combined to produce
a charge ratio (.+-.) of about 1:1 to about 20:1, preferably in a
ratio of about 1:1 to about 12:1, and more preferably in a ratio of
about 2:1 to about 6:1. Additionally, the overall concentration of
siNA in solution will typically be from about 25 .mu.g/mL to about
1 mg/mL, preferably from about 25 .mu.g/mL to about 500 .mu.g/mL,
and more preferably from about 100 .mu.g/mL to about 250 .mu.g/mL.
The combination of the molecules of interest and cationic lipids in
detergent solution is kept, typically at room temperature, for a
period of time which is sufficient for the coated complexes to
form. Alternatively, the molecules of interest and cationic lipids
can be combined in the detergent solution and warmed to
temperatures of up to about 37.degree. C. For molecules (e.g.,
certain polynucleotides herein) which are particularly sensitive to
temperature, the coated complexes can be formed at lower
temperatures, typically down to about 4.degree. C.
[0209] In one embodiment, the siNA to lipid ratios (mass/mass
ratios) in a formed formulated molecular composition range from
about 0.01 to about 0.08. The ratio of the starting materials also
falls within this range because the purification step typically
removes the unencapsulated siNA as well as the empty liposomes. In
another embodiment, the formulated siNA composition preparation
uses about 400 .mu.g siNA per 10 mg total lipid or a siNA to lipid
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 siNA. A
formulated molecular composition of the invention is developed to
target specific organs, tissues, or cell types. In one embodiment,
a formulated molecular composition of the invention is developed to
target the liver or hepatocytes. Ratios of the various components
of the formulated molecular composition are adjusted to target
specific organs, tissues, or cell types.
[0210] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to a
cell or cells in a subject or organism, comprising administering a
formulated molecular composition of the invention under conditions
suitable for delivery of the biologically active molecule component
of the formulated molecular composition to the cell or cells of the
subject or organism. In one embodiment, the formulated molecular
composition is contacted with the cell or cells of the subject or
organism as is generally known in the art, such as via parental
administration (e.g., intravenous, intramuscular, subcutaneous
administration) or pulmonary administration of the formulated
molecular composition with or without excipients to facilitate the
administration.
[0211] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to liver
or liver cells (e.g., hepatocytes) in a subject or organism,
comprising administering a formulated molecular composition of the
invention under conditions suitable for delivery of the
biologically active molecule component of the formulated molecular
composition to the liver or liver cells (e.g., hepatocytes) of the
subject or organism. In one embodiment, the formulated molecular
composition is contacted with the liver or liver cells of the
subject or organism as is generally known in the art, such as via
parental administration (e.g., intravenous, intramuscular,
subcutaneous administration) or local administration (e.g., direct
injection, portal vein injection, catheterization, stenting etc.)
of the formulated molecular composition with or without excipients
to facilitate the administration.
[0212] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to
kidney or kidney cells in a subject or organism, comprising
administering a formulated molecular composition of the invention
under conditions suitable for delivery of the biologically active
molecule component of the formulated molecular composition to the
kidney or kidney cells of the subject or organism. In one
embodiment, the formulated molecular composition is contacted with
the kidney or kidney cells of the subject or organism as is
generally known in the art, such as via parental administration
(e.g., intravenous, intramuscular, subcutaneous administration) or
local administration (e.g., direct injection, catheterization,
stenting etc.) of the formulated molecular composition with or
without excipients to facilitate the administration.
[0213] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to tumor
or tumor cells in a subject or organism, comprising administering a
formulated molecular composition of the invention under conditions
suitable for delivery of the biologically active molecule component
of the formulated molecular composition to the tumor or tumor cells
of the subject or organism. In one embodiment, the formulated
molecular composition is contacted with the tumor or tumor cells of
the subject or organism as is generally known in the art, such as
via parental administration (e.g., intravenous, intramuscular,
subcutaneous administration) or local administration (e.g., direct
injection, catheterization, stenting etc.) of the formulated
molecular composition with or without excipients to facilitate the
administration.
[0214] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to CNS
or CNS cells (e.g., brain, spinal cord) in a subject or organism,
comprising administering a formulated molecular composition of the
invention under conditions suitable for delivery of the
biologically active molecule component of the formulated molecular
composition to the CNS or CNS cells of the subject or organism. In
one embodiment, the formulated molecular composition is contacted
with the CNS or CNS cells of the subject or organism as is
generally known in the art, such as via parental administration
(e.g., intravenous, intramuscular, subcutaneous administration) or
local administration (e.g., direct injection, catheterization,
stenting etc.) of the formulated molecular composition with or
without excipients to facilitate the administration.
[0215] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to lung
or lung cells in a subject or organism, comprising administering a
formulated molecular composition of the invention under conditions
suitable for delivery of the biologically active molecule component
of the formulated molecular composition to the lung or lung cells
of the subject or organism. In one embodiment, the formulated
molecular composition is contacted with the lung or lung cells of
the subject or organism as is generally known in the art, such as
via parental administration (e.g., intravenous, intramuscular,
subcutaneous administration) or local administration (e.g.,
pulmonary administration directly to lung tissues and cells) of the
formulated molecular composition with or without excipients to
facilitate the administration.
[0216] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to
vascular or vascular cells in a subject or organism, comprising
administering a formulated molecular composition of the invention
under conditions suitable for delivery of the biologically active
molecule component of the formulated molecular composition to the
vascular or vascular cells of the subject or organism. In one
embodiment, the formulated molecular composition is contacted with
the vascular or vascular cells of the subject or organism as is
generally known in the art, such as via parental administration
(e.g., intravenous, intramuscular, subcutaneous administration) or
local administration (e.g., clamping, catheterization, stenting
etc.) of the formulated molecular composition with or without
excipients to facilitate the administration.
[0217] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to skin
or skin cells (e.g., dermis or dermis cells, follicle or follicular
cells) in a subject or organism, comprising administering a
formulated molecular composition of the invention under conditions
suitable for delivery of the biologically active molecule component
of the formulated molecular composition to the skin or skin cells
of the subject or organism. In one embodiment, the formulated
molecular composition is contacted with the skin or skin cells of
the subject or organism as is generally known in the art, such as
via parental administration (e.g., intravenous, intramuscular,
subcutaneous administration) or local administration (e.g., direct
dermal application, iontophoresis etc.) of the formulated molecular
composition with or without excipients to facilitate the
administration.
[0218] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to the
eye or ocular cells (e.g., macula, fovea, cornea, retina etc.) in a
subject or organism, comprising administering a formulated
molecular composition of the invention under conditions suitable
for delivery of the biologically active molecule component of the
formulated molecular composition to the eye or ocular cells of the
subject or organism. In one embodiment, the formulated molecular
composition is contacted with the eye or ocular cells of the
subject or organism as is generally known in the art, such as via
parental administration (e.g., intravenous, intramuscular,
subcutaneous administration) or local administration (e.g., direct
injection, intraocular injection, periocular injection,
iontophoresis, use of eyedrops, inplants etc.) of the formulated
molecular composition with or without excipients to facilitate the
administration.
[0219] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule to the
ear or cells of the ear (e.g., inner ear, middle ear, outer ear) in
a subject or organism, comprising administering a formulated
molecular composition of the invention under conditions suitable
for delivery of the biologically active molecule component of the
formulated molecular composition to the ear or ear cells of the
subject or organism. In one embodiment, the administration
comprises methods and devices as described in U.S. Pat. Nos.
5,421,818, 5,476,446, 5,474,529, 6,045,528, 6,440,102, 6,685,697,
6,120,484; and 5,572,594; all incorporated by reference in their
entireties herein and the teachings of Silverstein, 1999, Ear Nose
Throat J., 78, 595-8, 600; and Jackson and Silverstein, 2002,
Otolaryngol Clin North Am., 35, 639-53, and adapted for use the
compositions of the invention.
[0220] In one embodiment, the invention features a formulated siNA
composition comprising a short interfering nucleic acid (siNA)
molecule that down-regulates expression of a target gene, wherein
said siNA molecule comprises about 15 to about 28 base pairs.
[0221] In one embodiment, the invention features a formulated siNA
composition comprising a double stranded short interfering nucleic
acid (siNA) molecule that directs cleavage of a target RNA via RNA
interference (RNAi), wherein the double stranded siNA molecule
comprises a first and a second strand, each strand of the siNA
molecule is about 18 to about 28 nucleotides in length, the first
strand of the siNA comprises nucleotide sequence having sufficient
complementarity to the target RNA for the siNA molecule to direct
cleavage of the target RNA via RNA interference, and the second
strand of said siNA molecule comprises nucleotide sequence that is
complementary to the first strand.
[0222] In one embodiment, the invention features a formulated siNA
composition comprising a double stranded short interfering nucleic
acid (siNA) molecule that directs cleavage of a target RNA via RNA
interference (RNAi), wherein the double stranded siNA molecule
comprises a first and a second strand, each strand of the siNA
molecule is about 18 to about 23 nucleotides in length, the first
strand of the siNA molecule comprises nucleotide sequence having
sufficient complementarity to the target RNA for the siNA molecule
to direct cleavage of the target RNA via RNA interference, and the
second strand of said siNA molecule comprises nucleotide sequence
that is complementary to the first strand.
[0223] In one embodiment, the invention features a formulated siNA
composition comprising a chemically synthesized double stranded
short interfering nucleic acid (siNA) molecule that directs
cleavage of a target RNA via RNA interference (RNAi), wherein each
strand of the siNA molecule is about 18 to about 28 nucleotides in
length; and one strand of the siNA molecule comprises nucleotide
sequence having sufficient complementarity to the target RNA for
the siNA molecule to direct cleavage of the target RNA via RNA
interference.
[0224] In one embodiment, the invention features a formulated siNA
composition comprising a chemically synthesized double stranded
short interfering nucleic acid (siNA) molecule that directs
cleavage of a target RNA via RNA interference (RNAi), wherein each
strand of the siNA molecule is about 18 to about 23 nucleotides in
length; and one strand of the siNA molecule comprises nucleotide
sequence having sufficient complementarity to the target RNA for
the siNA molecule to direct cleavage of the target RNA via RNA
interference.
[0225] In one embodiment, the invention features a formulated siNA
composition comprising a siNA molecule that down-regulates
expression of a target gene, for example, wherein the target gene
comprises target encoding sequence. In one embodiment, the
invention features a siNA molecule that down-regulates expression
of a target gene, for example, wherein the target gene comprises
target non-coding sequence or regulatory elements involved in
target gene expression.
[0226] In one embodiment, a siNA of the invention is used to
inhibit the expression of target genes or a target gene family,
wherein the genes or gene family sequences share sequence homology.
Such homologous sequences can be identified as is known in the art,
for example using sequence alignments. siNA molecules can be
designed to target such homologous sequences, for example using
perfectly complementary sequences or by incorporating non-canonical
base pairs, for example mismatches and/or wobble base pairs that
can provide additional target sequences. In instances where
mismatches are identified, non-canonical base pairs (for example,
mismatches and/or wobble bases) can be used to generate siNA
molecules that target more than one gene sequence. In a
non-limiting example, non-canonical base pairs such as UU and CC
base pairs are used to generate siNA molecules that are capable of
targeting sequences for differing targets that share sequence
homology. As such, one advantage of using siNAs of the invention is
that a single siNA can be designed to include nucleic acid sequence
that is complementary to the nucleotide sequence that is conserved
between the homologous genes. In this approach, a single siNA can
be used to inhibit expression of more than one gene instead of
using more than one siNA molecule to target the different
genes.
[0227] In one embodiment, the invention features a formulated siNA
composition comprising a siNA molecule having RNAi activity against
a target RNA, wherein the siNA molecule comprises a sequence
complementary to any RNA having target encoding sequence. Examples
of siNA molecules suitable for the formulations described herein
are provided in International Application Ser. Nos. 04/106390 (WO
05/19453), which is hereby incorporated by reference in its
entirety. Chemical modifications as described in PCT/US 2004/106390
(WO 05/19453), U.S. Ser. No. 10/444,853, filed May 23, 2003 U.S.
Ser. No. 10/923,536 filed Aug. 20, 2004, U.S. Ser. No. 11/234,730,
filed Sep. 23, 2005 or U.S. Ser. No. 11/299,254, filed Dec. 8,
2005, all incorporated by reference in their entireties herein, or
otherwise described herein can be applied to any siNA construct of
the invention. In another embodiment, a siNA molecule of the
invention includes a nucleotide sequence that can interact with
nucleotide sequence of a target gene and thereby mediate silencing
of target gene expression, for example, wherein the siNA mediates
regulation of target gene expression by cellular processes that
modulate the chromatin structure or methylation patterns of the
target gene and prevent transcription of the target gene.
[0228] In one embodiment, siNA molecules of the invention are used
to down regulate or inhibit the expression of target proteins
arising from target haplotype polymorphisms that are associated
with a disease or condition (e.g. alopecia, hair loss, and/or
atrichia). Analysis of target genes, or target protein or RNA
levels can be used to identify subjects with such polymorphisms or
those subjects who are at risk of developing traits, conditions, or
diseases described herein. These subjects are amenable to
treatment, for example, treatment with siNA molecules of the
invention and any other composition useful in treating diseases
related to target gene expression. As such, analysis of target
protein or RNA levels can be used to determine treatment type and
the course of therapy in treating a subject. Monitoring of target
protein or RNA levels can be used to predict treatment outcome and
to determine the efficacy of compounds and compositions that
modulate the level and/or activity of certain target proteins
associated with a trait, condition, or disease.
[0229] In one embodiment, a siNA molecule of the invention
comprises an antisense strand comprising a nucleotide sequence that
is complementary to a nucleotide sequence or a portion thereof
encoding a target protein. The siNA further comprises a sense
strand, wherein said sense strand comprises a nucleotide sequence
of a target gene or a portion thereof.
[0230] In another embodiment, a siNA of the invention comprises an
antisense region comprising a nucleotide sequence that is
complementary to a nucleotide sequence encoding a target protein or
a portion thereof. The siNA molecule further comprises a sense
region, wherein said sense region comprises a nucleotide sequence
of a target gene or a portion thereof.
[0231] In another embodiment, a siNA of the invention comprises a
nucleotide sequence in the antisense region of the siNA molecule
that is complementary to a nucleotide sequence or portion of
sequence of a target gene. In another embodiment, a siNA of the
invention comprises a region, for example, the antisense region of
the siNA construct that is complementary to a sequence comprising a
target gene sequence or a portion thereof.
[0232] In one embodiment, a siNA molecule of the invention
comprises an antisense strand having about 15 to about 30 (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30) nucleotides, wherein the antisense strand is complementary
to a RNA sequence or a portion thereof encoding a target protein,
and wherein said siNA further comprises a sense strand having about
15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30) nucleotides, and wherein said sense
strand and said antisense strand are distinct nucleotide sequences
where at least about 15 nucleotides in each strand are
complementary to the other strand.
[0233] In another embodiment, a siNA molecule of the invention
comprises an antisense region having about 15 to about 30 (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30) nucleotides, wherein the antisense region is complementary
to a RNA sequence encoding a target protein, and wherein said siNA
further comprises a sense region having about 15 to about 30 (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30) nucleotides, wherein said sense region and said antisense
region are comprised in a linear molecule where the sense region
comprises at least about 15 nucleotides that are complementary to
the antisense region.
[0234] In one embodiment, a siNA molecule of the invention has RNAi
activity that modulates expression of RNA encoded by a target gene.
Because target genes can share some degree of sequence homology
with each other, siNA molecules can be designed to target a class
of target genes or alternately specific target genes (e.g.,
polymorphic variants) by selecting sequences that are either shared
amongst different targets or alternatively that are unique for a
specific target. Therefore, in one embodiment, the siNA molecule
can be designed to target conserved regions of target RNA sequences
having homology among several target gene variants so as to target
a class of target genes with one siNA molecule. Accordingly, in one
embodiment, the siNA molecule of the invention modulates the
expression of one or both target alleles in a subject. In another
embodiment, the siNA molecule can be designed to target a sequence
that is unique to a specific target RNA sequence (e.g., a single
target allele or target single nucleotide polymorphism (SNP)) due
to the high degree of specificity that the siNA molecule requires
to mediate RNAi activity.
[0235] In one embodiment, a siNA molecule of the invention is
double-stranded. In another embodiment, the siNA molecules of the
invention consist of duplex nucleic acid molecules containing about
15 to about 30 base pairs between oligonucleotides comprising about
15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment,
siNA molecules of the invention comprise duplex nucleic acid
molecules with overhanging ends of about 1 to about 3 (e.g., about
1, 2, or 3) nucleotides, for example, about 21-nucleotide duplexes
with about 19 base pairs and 3'-terminal mononucleotide,
dinucleotide, or trinucleotide overhangs. In yet another
embodiment, siNA molecules of the invention comprise duplex nucleic
acid molecules with blunt ends, where both ends are blunt, or
alternatively, where one of the ends is blunt.
[0236] In one embodiment, siNA molecules of the invention have
specificity for nucleic acid molecules expressing target proteins,
such as RNA encoding a target protein. In one embodiment, a siNA
molecule of the invention is RNA based (e.g., a siNA comprising
2'-OH nucleotides) and includes one or more chemical modifications,
such as those described herein. Non-limiting examples of such
chemical modifications include without limitation phosphorothioate
internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl
ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal
base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides,
and terminal glyceryl and/or inverted deoxy abasic residue
incorporation. These chemical modifications, when used in various
siNA constructs, (e.g., RNA based siNA constructs), are shown to
preserve RNAi activity in cells while at the same time,
dramatically increasing the serum stability of these compounds.
Furthermore, contrary to the data published by Parrish et al.,
supra, applicant demonstrates that multiple (greater than one)
phosphorothioate substitutions are well-tolerated and confer
substantial increases in serum stability for modified siNA
constructs.
[0237] In one embodiment, a siNA molecule of the invention
comprises modified nucleotides while maintaining the ability to
mediate RNAi. The modified nucleotides can be used to improve in
vitro or in vivo characteristics such as stability, activity,
and/or bioavailability. For example, a siNA molecule of the
invention can comprise modified nucleotides as a percentage of the
total number of nucleotides present in the siNA molecule. As such,
a siNA molecule of the invention can generally comprise about 5% to
about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or 100% modified nucleotides). The actual percentage of
modified nucleotides present in a given siNA molecule will depend
on the total number of nucleotides present in the siNA. If the siNA
molecule is single stranded, the percent modification can be based
upon the total number of nucleotides present in the single stranded
siNA molecules. Likewise, if the siNA molecule is double stranded,
the percent modification can be based upon the total number of
nucleotides present in the sense strand, antisense strand, or both
the sense and antisense strands.
[0238] One aspect of the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene. In one embodiment, the double stranded siNA molecule
comprises one or more chemical modifications and each strand of the
double-stranded siNA is about 21 nucleotides long. In one
embodiment, the double-stranded siNA molecule does not contain any
ribonucleotides. In another embodiment, the double-stranded siNA
molecule comprises one or more ribonucleotides. In one embodiment,
each strand of the double-stranded siNA molecule independently
comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein
each strand comprises about 15 to about 30 (e.g., about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides
that are complementary to the nucleotides of the other strand. In
one embodiment, one of the strands of the double-stranded siNA
molecule comprises a nucleotide sequence that is complementary to a
nucleotide sequence or a portion thereof of the target gene, and
the second strand of the double-stranded siNA molecule comprises a
nucleotide sequence substantially similar to the nucleotide
sequence of the target gene or a portion thereof.
[0239] In another embodiment, the invention features a formulated
siNA composition comprising a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a
target gene comprising an antisense region, wherein the antisense
region comprises a nucleotide sequence that is complementary to a
nucleotide sequence of the target gene or a portion thereof, and a
sense region, wherein the sense region comprises a nucleotide
sequence substantially similar to the nucleotide sequence of the
target gene or a portion thereof. In one embodiment, the antisense
region and the sense region independently comprise about 15 to
about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, wherein the antisense region
comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are
complementary to nucleotides of the sense region.
[0240] In another embodiment, the invention features a formulated
siNA composition comprising a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a
target gene comprising a sense region and an antisense region,
wherein the antisense region comprises a nucleotide sequence that
is complementary to a nucleotide sequence of RNA encoded by the
target gene or a portion thereof and the sense region comprises a
nucleotide sequence that is complementary to the antisense
region.
[0241] In one embodiment, a siNA molecule of the invention
comprises blunt ends, i.e., ends that do not include any
overhanging nucleotides. For example, a siNA molecule comprising
modifications described in U.S. Ser. No. 10/444,853, filed May 23,
2003, U.S. Ser. No. 10/923,536 filed Aug. 20, 2004, or U.S. Ser.
No. 11/234,730, filed Sep. 23, 2005, all incorporated by reference
in their entireties herein, or any combination thereof and/or any
length described herein can comprise blunt ends or ends with no
overhanging nucleotides.
[0242] In one embodiment, any siNA molecule of the invention can
comprise one or more blunt ends, i.e. where a blunt end does not
have any overhanging nucleotides. In one embodiment, the blunt
ended siNA molecule has a number of base pairs equal to the number
of nucleotides present in each strand of the siNA molecule. In
another embodiment, the siNA molecule comprises one blunt end, for
example wherein the 5'-end of the antisense strand and the 3'-end
of the sense strand do not have any overhanging nucleotides. In
another example, the siNA molecule comprises one blunt end, for
example wherein the 3'-end of the antisense strand and the 5'-end
of the sense strand do not have any overhanging nucleotides. In
another example, a siNA molecule comprises two blunt ends, for
example wherein the 3'-end of the antisense strand and the 5'-end
of the sense strand as well as the 5'-end of the antisense strand
and 3'-end of the sense strand do not have any overhanging
nucleotides. A blunt ended siNA molecule can comprise, for example,
from about 15 to about 30 nucleotides (e.g., about 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).
Other nucleotides present in a blunt ended siNA molecule can
comprise, for example, mismatches, bulges, loops, or wobble base
pairs to modulate the activity of the siNA molecule to mediate RNA
interference.
[0243] By "blunt ends" is meant symmetric termini, or termini of a
double stranded siNA molecule having no overhanging nucleotides.
The two strands of a double stranded siNA molecule align with each
other without over-hanging nucleotides at the termini. For example,
a blunt ended siNA construct comprises terminal nucleotides that
are complementary between the sense and antisense regions of the
siNA molecule.
[0244] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene, wherein the siNA molecule is assembled from two separate
oligonucleotide fragments wherein one fragment comprises the sense
region and the second fragment comprises the antisense region of
the siNA molecule. The sense region can be connected to the
antisense region via a linker molecule, such as a polynucleotide
linker or a non-nucleotide linker.
[0245] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene, wherein the siNA molecule comprises about 15 to about 30
(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30) base pairs, and wherein each strand of the siNA molecule
comprises one or more chemical modifications. In another
embodiment, one of the strands of the double-stranded siNA molecule
comprises a nucleotide sequence that is complementary to a
nucleotide sequence of a target gene or a portion thereof, and the
second strand of the double-stranded siNA molecule comprises a
nucleotide sequence substantially similar to the nucleotide
sequence or a portion thereof of the target gene. In another
embodiment, one of the strands of the double-stranded siNA molecule
comprises a nucleotide sequence that is complementary to a
nucleotide sequence of a target gene or portion thereof, and the
second strand of the double-stranded siNA molecule comprises a
nucleotide sequence substantially similar to the nucleotide
sequence or portion thereof of the target gene. In another
embodiment, each strand of the siNA molecule comprises about 15 to
about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, and each strand comprises at
least about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are
complementary to the nucleotides of the other strand.
[0246] In any of the embodiments described herein, a siNA molecule
of the invention can comprise no ribonucleotides. Alternatively, a
siNA molecule of the invention can comprise one or more
ribonucleotides.
[0247] In one embodiment, a siNA molecule of the invention
comprises an antisense region comprising a nucleotide sequence that
is complementary to a nucleotide sequence of a target gene or a
portion thereof, and the siNA further comprises a sense region
comprising a nucleotide sequence substantially similar to the
nucleotide sequence of the target gene or a portion thereof. In
another embodiment, the antisense region and the sense region each
comprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides and the
antisense region comprises at least about 15 to about 30 (e.g.
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30) nucleotides that are complementary to nucleotides of the
sense region. The target gene can comprise, for example, sequences
referred to by Genbank Accession Nos. in PCT Publication No. WO
03/74654, serial No. PCT/US03/05028 or U.S. Ser. No. 10/923,536. In
another embodiment, the siNA is a double stranded nucleic acid
molecule, where each of the two strands of the siNA molecule
independently comprise about 15 to about 40 (e.g. about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34,
35, 36, 37, 38, 39, or 40) nucleotides, and where one of the
strands of the siNA molecule comprises at least about 15 (e.g.
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more)
nucleotides that are complementary to the nucleic acid sequence of
the target gene or a portion thereof.
[0248] In one embodiment, a siNA molecule of the invention
comprises a sense region and an antisense region, wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by a target
gene, or a portion thereof, and the sense region comprises a
nucleotide sequence that is complementary to the antisense region.
In one embodiment, the siNA molecule is assembled from two separate
oligonucleotide fragments, wherein one fragment comprises the sense
region and the second fragment comprises the antisense region of
the siNA molecule. In another embodiment, the sense region is
connected to the antisense region via a linker molecule. In another
embodiment, the sense region is connected to the antisense region
via a linker molecule, such as a nucleotide or non-nucleotide
linker. The target gene can comprise, for example, sequences
referred to in PCT Publication No. WO 03/74654, serial No.
PCT/US03/05028 or U.S. Ser. No. 10/923,536 or otherwise known in
the art.
[0249] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene comprising a sense region and an antisense region, wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by the target
gene or a portion thereof and the sense region comprises a
nucleotide sequence that is complementary to the antisense region,
and wherein the siNA molecule has one or more modified pyrimidine
and/or purine nucleotides. In one embodiment, the pyrimidine
nucleotides in the sense region are 2'-O-methyl pyrimidine
nucleotides or 2'-deoxy-2'-fluoro pyrimidine nucleotides and the
purine nucleotides present in the sense region are 2'-deoxy purine
nucleotides. In another embodiment, the pyrimidine nucleotides in
the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and
the purine nucleotides present in the sense region are 2'-O-methyl
purine nucleotides. In another embodiment, the pyrimidine
nucleotides in the sense region are 2'-deoxy-2'-fluoro pyrimidine
nucleotides and the purine nucleotides present in the sense region
are 2'-deoxy purine nucleotides. In one embodiment, the pyrimidine
nucleotides in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine nucleotides and the purine nucleotides present in the
antisense region are 2'-O-methyl or 2'-deoxy purine nucleotides. In
another embodiment of any of the above-described siNA molecules,
any nucleotides present in a non-complementary region of the sense
strand (e.g. overhang region) are 2'-deoxy nucleotides.
[0250] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene, wherein the siNA molecule is assembled from two separate
oligonucleotide fragments wherein one fragment comprises the sense
region and the second fragment comprises the antisense region of
the siNA molecule, and wherein the fragment comprising the sense
region includes a terminal cap moiety at the 5'-end, the 3'-end, or
both of the 5' and 3' ends of the fragment. In one embodiment, the
terminal cap moiety is an inverted deoxy abasic moiety or glyceryl
moiety. In one embodiment, each of the two fragments of the siNA
molecule independently comprise about 15 to about 30 (e.g. about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides. In another embodiment, each of the two fragments of
the siNA molecule independently comprise about 15 to about 40 (e.g.
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a
non-limiting example, each of the two fragments of the siNA
molecule comprises about 21 nucleotides.
[0251] In one embodiment, the invention features a formulated siNA
composition comprising a siNA molecule comprising at least one
modified nucleotide, wherein the modified nucleotide is a
2'-deoxy-2'-fluoro nucleotide. The siNA can be, for example, about
15 to about 40 nucleotides in length. In one embodiment, all
pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
pyrimidine nucleotides. In one embodiment, the modified nucleotides
in the siNA include at least one 2'-deoxy-2'-fluoro cytidine or
2'-deoxy-2'-fluoro uridine nucleotide. In another embodiment, the
modified nucleotides in the siNA include at least one 2'-fluoro
cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotides.
In one embodiment, all uridine nucleotides present in the siNA are
2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all
cytidine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
cytidine nucleotides. In one embodiment, all adenosine nucleotides
present in the siNA are 2'-deoxy-2'-fluoro adenosine nucleotides.
In one embodiment, all guanosine nucleotides present in the siNA
are 2'-deoxy-2'-fluoro guanosine nucleotides. The siNA can further
comprise at least one modified internucleotidic linkage, such as
phosphorothioate linkage. In one embodiment, the
2'-deoxy-2'-fluoronucleotides are present at specifically selected
locations in the siNA that are sensitive to cleavage by
ribonucleases, such as locations having pyrimidine nucleotides.
[0252] In one embodiment, the invention features a method of
increasing the stability of a siNA molecule of the invention
against cleavage by ribonucleases comprising introducing at least
one modified nucleotide into the siNA molecule, wherein the
modified nucleotide is a 2'-deoxy-2'-fluoro nucleotide. In one
embodiment, all pyrimidine nucleotides present in the siNA are
2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the
modified nucleotides in the siNA include at least one
2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine
nucleotide. In another embodiment, the modified nucleotides in the
siNA include at least one 2'-fluoro cytidine and at least one
2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all
uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
uridine nucleotides. In one embodiment, all cytidine nucleotides
present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In
one embodiment, all adenosine nucleotides present in the siNA are
2'-deoxy-2'-fluoro adenosine nucleotides. In one embodiment, all
guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
guanosine nucleotides. The siNA can further comprise at least one
modified internucleotidic linkage, such as phosphorothioate
linkage. In one embodiment, the 2'-deoxy-2'-fluoronucleotides are
present at specifically selected locations in the siNA that are
sensitive to cleavage by ribonucleases, such as locations having
pyrimidine nucleotides.
[0253] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene comprising a sense region and an antisense region, wherein the
antisense region comprises a nucleotide sequence that is
complementary to a nucleotide sequence of RNA encoded by the target
gene or a portion thereof and the sense region comprises a
nucleotide sequence that is complementary to the antisense region,
and wherein the purine nucleotides present in the antisense region
comprise 2'-deoxy-purine nucleotides. In an alternative embodiment,
the purine nucleotides present in the antisense region comprise
2'-O-methyl purine nucleotides. In either of the above embodiments,
the antisense region can comprise a phosphorothioate
internucleotide linkage at the 3' end of the antisense region.
Alternatively, in either of the above embodiments, the antisense
region can comprise a glyceryl modification at the 3' end of the
antisense region. In another embodiment of any of the
above-described siNA molecules, any nucleotides present in a
non-complementary region of the antisense strand (e.g. overhang
region) are 2'-deoxy nucleotides.
[0254] In one embodiment, the antisense region of a siNA molecule
of the invention comprises sequence complementary to a portion of a
target transcript having sequence unique to a particular target
disease related allele, such as sequence comprising a single
nucleotide polymorphism (SNP) associated with the disease specific
allele. As such, the antisense region of a siNA molecule of the
invention can comprise sequence complementary to sequences that are
unique to a particular allele to provide specificity in mediating
selective RNAi against the disease, condition, or trait related
allele.
[0255] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that down-regulates expression of a target
gene, wherein the siNA molecule is assembled from two separate
oligonucleotide fragments wherein one fragment comprises the sense
region and the second fragment comprises the antisense region of
the siNA molecule. In another embodiment, the siNA molecule is a
double stranded nucleic acid molecule, where each strand is about
21 nucleotides long and where about 19 nucleotides of each fragment
of the siNA molecule are base-paired to the complementary
nucleotides of the other fragment of the siNA molecule, wherein at
least two 3' terminal nucleotides of each fragment of the siNA
molecule are not base-paired to the nucleotides of the other
fragment of the siNA molecule. In another embodiment, the siNA
molecule is a double stranded nucleic acid molecule, where each
strand is about 19 nucleotide long and where the nucleotides of
each fragment of the siNA molecule are base-paired to the
complementary nucleotides of the other fragment of the siNA
molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19)
base pairs, wherein one or both ends of the siNA molecule are blunt
ends. In one embodiment, each of the two 3' terminal nucleotides of
each fragment of the siNA molecule is a 2'-deoxy-pyrimidine
nucleotide, such as a 2'-deoxy-thymidine. In another embodiment,
all nucleotides of each fragment of the siNA molecule are
base-paired to the complementary nucleotides of the other fragment
of the siNA molecule. In another embodiment, the siNA molecule is a
double stranded nucleic acid molecule of about 19 to about 25 base
pairs having a sense region and an antisense region, where about 19
nucleotides of the antisense region are base-paired to the
nucleotide sequence or a portion thereof of the RNA encoded by the
target gene. In another embodiment, about 21 nucleotides of the
antisense region are base-paired to the nucleotide sequence or a
portion thereof of the RNA encoded by the target gene. In any of
the above embodiments, the 5'-end of the fragment comprising said
antisense region can optionally include a phosphate group.
[0256] In any of the embodiments described herein, a siNA molecule
of the invention can comprise one or more of the stabilization
chemistries shown in Table I or described in PCT/US 2004/106390 (WO
05/19453), U.S. Ser. No. 10/444,853, filed May 23, 2003 U.S. Ser.
No. 10/923,536 filed Aug. 20, 2004, U.S. Ser. No. 11/234,730, filed
Sep. 23, 2005 or U.S. Ser. No. 11/299,254, filed Dec. 8, 2005, all
incorporated by reference in their entireties herein.
[0257] In one embodiment, the invention features a formulated siNA
composition comprising a double-stranded short interfering nucleic
acid (siNA) molecule that inhibits the expression of a target RNA
sequence (e.g., wherein said target RNA sequence is encoded by a
target gene involved in the target pathway), wherein the siNA
molecule does not contain any ribonucleotides and wherein each
strand of the double-stranded siNA molecule is about 15 to about 30
nucleotides. In one embodiment, the siNA molecule is 21 nucleotides
in length. Examples of non-ribonucleotide containing siNA
constructs are combinations of stabilization chemistries described
in PCT/US 2004/106390 (WO 05/19453), U.S. Ser. No. 10/444,853,
filed May 23, 2003 U.S. Ser. No. 10/923,536 filed Aug. 20, 2004,
U.S. Ser. No. 11/234,730, filed Sep. 23, 2005 or U.S. Ser. No.
11/299,254, filed Dec. 8, 2005, all incorporated by reference in
their entireties herein.
[0258] In one embodiment, the invention features a formulated siNA
composition comprising a chemically synthesized double stranded RNA
molecule that directs cleavage of a target RNA via RNA
interference, wherein each strand of said RNA molecule is about 15
to about 30 nucleotides in length; one strand of the RNA molecule
comprises nucleotide sequence having sufficient complementarity to
the target RNA for the RNA molecule to direct cleavage of the
target RNA via RNA interference; and wherein at least one strand of
the RNA molecule optionally comprises one or more chemically
modified nucleotides described herein, such as without limitation
deoxynucleotides, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro
nucleotides, 2'-O-methoxyethyl nucleotides etc.
[0259] In one embodiment, the invention features a composition
comprising a formulated siNA composition of the invention in a
pharmaceutically acceptable carrier or diluent.
[0260] In one embodiment, the invention features a double-stranded
short interfering nucleic acid (siNA) molecule that inhibits the
expression of a target RNA sequence, wherein the siNA molecule does
not contain any ribonucleotides and wherein each strand of the
double-stranded siNA molecule is about 15 to about 30 nucleotides.
In one embodiment, the siNA molecule is 21 nucleotides in length.
Examples of non-ribonucleotide containing siNA constructs are
combinations of stabilization chemistries shown in Table I in any
combination of Sense/Antisense chemistries, such as Stab 7/8, Stab
7/11, Stab 8/8, Stab 18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab
18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab
18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having
Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, 20, or 32 sense or
antisense strands or any combination thereof). Herein, numeric Stab
chemistries can include both 2'-fluoro and 2'-OCF3 versions of the
chemistries shown in Table I. For example, "Stab 7/8" refers to
both Stab 7/8 and Stab 7F/8F etc. In one embodiment, the invention
features a chemically synthesized double stranded RNA molecule that
directs cleavage of a target RNA via RNA interference, wherein each
strand of said RNA molecule is about 15 to about 30 nucleotides in
length; one strand of the RNA molecule comprises nucleotide
sequence having sufficient complementarity to the target RNA for
the RNA molecule to direct cleavage of the target RNA via RNA
interference; and wherein at least one strand of the RNA molecule
optionally comprises one or more chemically modified nucleotides
described herein, such as without limitation deoxynucleotides,
2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides,
2'-O-methoxyethyl nucleotides, 4'-thio nucleotides,
2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy
nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides, etc.
[0261] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) nucleotides comprising a backbone modified internucleotide
linkage having Formula I:
##STR00052##
[0262] wherein each R1 and R2 is independently any nucleotide,
non-nucleotide, or polynucleotide which can be naturally-occurring
or chemically-modified, each X and Y is independently O, S, N,
alkyl, or substituted alkyl, each Z and W is independently O, S, N,
alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or
acetyl and wherein W, X, Y, and Z are optionally not all O. In
another embodiment, a backbone modification of the invention
comprises a phosphonoacetate and/or thiophosphonoacetate
internucleotide linkage (see for example Sheehan et al., 2003,
Nucleic Acids Research, 31, 4109-4118).
[0263] The chemically-modified internucleotide linkages having
Formula I, for example, wherein any Z, W, X, and/or Y independently
comprises a sulphur atom, can be present in one or both
oligonucleotide strands of the siNA duplex, for example, in the
sense strand, the antisense strand, or both strands. The siNA
molecules of the invention can comprise one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modified
internucleotide linkages having Formula I at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the sense strand, the
antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more
(e.g., about 1, 2, 3, 4, 5, or more) chemically-modified
internucleotide linkages having Formula I at the 5'-end of the
sense strand, the antisense strand, or both strands. In another
non-limiting example, an exemplary siNA molecule of the invention
can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) pyrimidine nucleotides with chemically-modified
internucleotide linkages having Formula I in the sense strand, the
antisense strand, or both strands. In yet another non-limiting
example, an exemplary siNA molecule of the invention can comprise
one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
purine nucleotides with chemically-modified internucleotide
linkages having Formula I in the sense strand, the antisense
strand, or both strands. In another embodiment, a siNA molecule of
the invention having internucleotide linkage(s) of Formula I also
comprises a chemically-modified nucleotide or non-nucleotide having
any of Formulae I-VII.
[0264] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) nucleotides or non-nucleotides having Formula II:
##STR00053##
wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl,
alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I or
II; R9 is O, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic
base such as adenine, guanine, uracil, cytosine, thymine,
2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other
non-naturally occurring base that can be complementary or
non-complementary to target RNA or a non-nucleosidic base such as
phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine,
pyridone, pyridinone, or any other non-naturally occurring
universal base that can be complementary or non-complementary to
target RNA. In one embodiment, R3 and/or R7 comprises a conjugate
moiety and a linker (e.g., a nucleotide or non-nucleotide linker as
described herein or otherwise known in the art). Non-limiting
examples of conjugate moieties include ligands for cellular
receptors, such as peptides derived from naturally occurring
protein ligands; protein localization sequences, including cellular
ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and
other co-factors, such as folate and N-acetylgalactosamine;
polymers, such as polyethyleneglycol (PEG); phospholipids;
cholesterol; steroids, and polyamines, such as PEI, spermine or
spermidine.
[0265] The chemically-modified nucleotide or non-nucleotide of
Formula II can be present in one or both oligonucleotide strands of
the siNA duplex, for example in the sense strand, the antisense
strand, or both strands. The siNA molecules of the invention can
comprise one or more chemically-modified nucleotides or
non-nucleotides of Formula H at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of the sense strand, the antisense strand, or
both strands. For example, an exemplary siNA molecule of the
invention can comprise about 1 to about 5 or more (e.g., about 1,
2, 3, 4, 5, or more) chemically-modified nucleotides or
non-nucleotides of Formula II at the 5'-end of the sense strand,
the antisense strand, or both strands. In anther non-limiting
example, an exemplary siNA molecule of the invention can comprise
about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more)
chemically-modified nucleotides or non-nucleotides of Formula II at
the 3'-end of the sense strand, the antisense strand, or both
strands.
[0266] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) nucleotides or non-nucleotides having Formula III:
##STR00054##
wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl,
alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I or
II; R9 is O, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic
base such as adenine, guanine, uracil, cytosine, thymine,
2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other
non-naturally occurring base that can be employed to be
complementary or non-complementary to target RNA or a
non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,
5-nitroindole, nebularine, pyridone, pyridinone, or any other
non-naturally occurring universal base that can be complementary or
non-complementary to target RNA. In one embodiment, R3 and/or R7
comprises a conjugate moiety and a linker (e.g., a nucleotide or
non-nucleotide linker as described herein or otherwise known in the
art). Non-limiting examples of conjugate moieties include ligands
for cellular receptors, such as peptides derived from naturally
occurring protein ligands; protein localization sequences,
including cellular ZIP code sequences; antibodies; nucleic acid
aptamers; vitamins and other co-factors, such as folate and
N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);
phospholipids; cholesterol; steroids, and polyamines, such as PEI,
spermine or spermidine.
[0267] The chemically-modified nucleotide or non-nucleotide of
Formula III can be present in one or both oligonucleotide strands
of the siNA duplex, for example, in the sense strand, the antisense
strand, or both strands. The siNA molecules of the invention can
comprise one or more chemically-modified nucleotides or
non-nucleotides of Formula III at the 3'-end, the 5'-end, or both
of the 3' and 5'-ends of the sense strand, the antisense strand, or
both strands. For example, an exemplary siNA molecule of the
invention can comprise about 1 to about 5 or more (e.g., about 1,
2, 3, 4, 5, or more) chemically-modified nucleotide(s) or
non-nucleotide(s) of Formula III at the 5'-end of the sense strand,
the antisense strand, or both strands. In anther non-limiting
example, an exemplary siNA molecule of the invention can comprise
about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more)
chemically-modified nucleotide or non-nucleotide of Formula III at
the 3'-end of the sense strand, the antisense strand, or both
strands.
[0268] In another embodiment, a siNA molecule of the invention
comprises a nucleotide having Formula II or III, wherein the
nucleotide having Formula II or III is in an inverted
configuration. For example, the nucleotide having Formula II or III
is connected to the siNA construct in a 3'-3', 3'-2', 2'-3', or
5'-5' configuration, such as at the 3'-end, the 5'-end, or both of
the 3' and 5'-ends of one or both siNA strands.
[0269] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises a 5'-terminal phosphate group having Formula IV:
##STR00055##
wherein each X and Y is independently O, S, N, alkyl, substituted
alkyl, or alkylhalo; wherein each Z and W is independently O, S, N,
alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl,
alkylhalo, or acetyl; and wherein W, X, Y and Z are not all O.
[0270] In one embodiment, the invention features a siNA molecule
having a 5'-terminal phosphate group having Formula IV on the
target-complementary strand, for example, a strand complementary to
a target RNA, wherein the siNA molecule comprises an all RNA siNA
molecule. In another embodiment, the invention features a siNA
molecule having a 5'-terminal phosphate group having Formula IV on
the target-complementary strand wherein the siNA molecule also
comprises about 1 to about 3 (e.g., about 1, 2, or 3) nucleotide
3'-terminal nucleotide overhangs having about 1 to about 4 (e.g.,
about 1, 2, 3, or 4) deoxyribonucleotides on the 3'-end of one or
both strands. In another embodiment, a 5'-terminal phosphate group
having Formula IV is present on the target-complementary strand of
a siNA molecule of the invention, for example a siNA molecule
having chemical modifications having any of Formulae I-VII.
[0271] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
capable of mediating RNA interference (RNAi) inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more phosphorothioate internucleotide linkages.
For example, in a non-limiting example, the invention features a
chemically-modified short interfering nucleic acid (siNA) having
about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate
internucleotide linkages in one siNA strand. In yet another
embodiment, the invention features a chemically-modified short
interfering nucleic acid (siNA) individually having about 1, 2, 3,
4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in
both siNA strands. The phosphorothioate internucleotide linkages
can be present in one or both oligonucleotide strands of the siNA
duplex, for example in the sense strand, the antisense strand, or
both strands. The siNA molecules of the invention can comprise one
or more phosphorothioate internucleotide linkages at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends of the sense strand, the
antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more
(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate
internucleotide linkages at the 5'-end of the sense strand, the
antisense strand, or both strands. In another non-limiting example,
an exemplary siNA molecule of the invention can comprise one or
more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
pyrimidine phosphorothioate internucleotide linkages in the sense
strand, the antisense strand, or both strands. In yet another
non-limiting example, an exemplary siNA molecule of the invention
can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) purine phosphorothioate internucleotide linkages in
the sense strand, the antisense strand, or both strands.
[0272] In one embodiment, the invention features a siNA molecule,
wherein the sense strand comprises one or more, for example, about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy and/or
about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more) universal base modified nucleotides, and optionally a
terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'-
and 5'-ends of the sense strand; and wherein the antisense strand
comprises about 1 to about 10 or more, specifically about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide
linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense
strand. In another embodiment, one or more, for example about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the
sense and/or antisense siNA strand are chemically-modified with
2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio
and/or 2'-deoxy-2'-fluoro nucleotides, with or without one or more,
for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more,
phosphorothioate internucleotide linkages and/or a terminal cap
molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends,
being present in the same or different strand.
[0273] In another embodiment, the invention features a siNA
molecule, wherein the sense strand comprises about 1 to about 5,
specifically about 1, 2, 3, 4, or 5 phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3,
4, 5, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more (e.g.,
about 1, 2, 3, 4, 5, or more) universal base modified nucleotides,
and optionally a terminal cap molecule at the 3-end, the 5'-end, or
both of the 3'- and 5'-ends of the sense strand; and wherein the
antisense strand comprises about 1 to about 5 or more, specifically
about 1, 2, 3, 4, 5, or more phosphorothioate internucleotide
linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense
strand. In another embodiment, one or more, for example about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the
sense and/or antisense siNA strand are chemically-modified with
2'-deoxy, 2'-O-methyl, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio
and/or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to
about 5 or more, for example about 1, 2, 3, 4, 5, or more
phosphorothioate internucleotide linkages and/or a terminal cap
molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-ends,
being present in the same or different strand.
[0274] In one embodiment, the invention features a siNA molecule,
wherein the antisense strand comprises one or more, for example,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate
internucleotide linkages, and/or about one or more (e.g., about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio
and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more) universal base modified nucleotides, and optionally a
terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'-
and 5'-ends of the sense strand; and wherein the antisense strand
comprises about 1 to about 10 or more, specifically about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide
linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy, 4'-thio and/or one or more (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base
modified nucleotides, and optionally a terminal cap molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends of the antisense
strand. In another embodiment, one or more, for example about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense
and/or antisense siNA strand are chemically-modified with 2'-deoxy,
2'-O-methyl, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'-fluoro
nucleotides, with or without one or more, for example, about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide
linkages and/or a terminal cap molecule at the 3'-end, the 5'-end,
or both of the 3' and 5'-ends, being present in the same or
different strand.
[0275] In another embodiment, the invention features a siNA
molecule, wherein the antisense strand comprises about 1 to about 5
or more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, 2'O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio
and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more) universal base modified nucleotides, and optionally a
terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'-
and 5'-ends of the sense strand; and wherein the antisense strand
comprises about 1 to about 5 or more, specifically about 1, 2, 3,
4, 5 or more phosphorothioate internucleotide linkages, and/or one
or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)
2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy, 4'-thio
and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more) universal base modified nucleotides, and optionally a
terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'-
and 5'-ends of the antisense strand. In another embodiment, one or
more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
pyrimidine nucleotides of the sense and/or antisense siNA strand
are chemically-modified with 2'-deoxy, 2'-O-methyl,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy, 4'-thio and/or 2'-deoxy-2'-fluoro
nucleotides, with or without about 1 to about 5, for example about
1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages
and/or a terminal cap molecule at the 3'-end, the 5'-end, or both
of the 3'- and 5'-ends, being present in the same or different
strand.
[0276] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
having about 1 to about 5 or more (specifically about 1, 2, 3, 4, 5
or more) phosphorothioate internucleotide linkages in each strand
of the siNA molecule.
[0277] In another embodiment, the invention features a siNA
molecule comprising 2'-5' internucleotide linkages. The 2'-5'
internucleotide linkage(s) can be at the 3'-end, the 5'-end, or
both of the 3'- and 5'-ends of one or both siNA sequence strands.
In addition, the 2'-5' internucleotide linkage(s) can be present at
various other positions within one or both siNA sequence strands,
for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more including
every internucleotide linkage of a pyrimidine nucleotide in one or
both strands of the siNA molecule can comprise a 2'-5'
internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more including every internucleotide linkage of a purine nucleotide
in one or both strands of the siNA molecule can comprise a 2'-5'
internucleotide linkage.
[0278] In another embodiment, a chemically-modified siNA molecule
of the invention comprises a duplex having two strands, one or both
of which can be chemically-modified, wherein each strand is
independently about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in
length, wherein the duplex has about 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
base pairs, and wherein the chemical modification comprises a
structure having any of Formulae I-VII. For example, an exemplary
chemically-modified siNA molecule of the invention comprises a
duplex having two strands, one or both of which can be
chemically-modified with a chemical modification having any of
Formulae I-VII or any combination thereof, wherein each strand
consists of about 21 nucleotides, each having a 2-nucleotide
3'-terminal nucleotide overhang, and wherein the duplex has about
19 base pairs. In another embodiment, a siNA molecule of the
invention comprises a single stranded hairpin structure, wherein
the siNA is about 36 to about 70 (e.g., about 36, 40, 45, 50, 55,
60, 65, or 70) nucleotides in length having about 15 to about 30
(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30) base pairs, and wherein the siNA can include a
chemical modification comprising a structure having any of Formulae
I-VII or any combination thereof. For example, an exemplary
chemically-modified siNA molecule of the invention comprises a
linear oligonucleotide having about 42 to about 50 (e.g., about 42,
43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is
chemically-modified with a chemical modification having any of
Formulae I-VII or any combination thereof, wherein the linear
oligonucleotide forms a hairpin structure having about 19 to about
21 (e.g., 19, 20, or 21) base pairs and a 2-nucleotide 3'-terminal
nucleotide overhang. In another embodiment, a linear hairpin siNA
molecule of the invention contains a stem loop motif, wherein the
loop portion of the siNA molecule is biodegradable. For example, a
linear hairpin siNA molecule of the invention is designed such that
degradation of the loop portion of the siNA molecule in vivo can
generate a double-stranded siNA molecule with 3'-terminal
overhangs, such as 3'-terminal nucleotide overhangs comprising
about 2 nucleotides.
[0279] In another embodiment, a siNA molecule of the invention
comprises a hairpin structure, wherein the siNA is about 25 to
about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50)
nucleotides in length having about 3 to about 25 (e.g., about 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25) base pairs, and wherein the siNA can include one or
more chemical modifications comprising a structure having any of
Formulae I-VII or any combination thereof. For example, an
exemplary chemically-modified siNA molecule of the invention
comprises a linear oligonucleotide having about 25 to about 35
(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)
nucleotides that is chemically-modified with one or more chemical
modifications having any of Formulae I-VII or any combination
thereof, wherein the linear oligonucleotide forms a hairpin
structure having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or
25) base pairs and a 5'-terminal phosphate group that can be
chemically modified as described herein (for example a 5'-terminal
phosphate group having Formula IV). In another embodiment, a linear
hairpin siNA molecule of the invention contains a stem loop motif,
wherein the loop portion of the siNA molecule is biodegradable. In
one embodiment, a linear hairpin siNA molecule of the invention
comprises a loop portion comprising a non-nucleotide linker.
[0280] In another embodiment, a siNA molecule of the invention
comprises an asymmetric hairpin structure, wherein the siNA is
about 25 to about 50 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
or 50) nucleotides in length having about 3 to about 25 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can
include one or more chemical modifications comprising a structure
having any of Formulae I-VII or any combination thereof. For
example, an exemplary chemically-modified siNA molecule of the
invention comprises a linear oligonucleotide having about 25 to
about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35) nucleotides that is chemically-modified with one or more
chemical modifications having any of Formulae I-VII or any
combination thereof, wherein the linear oligonucleotide forms an
asymmetric hairpin structure having about 3 to about 25 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25) base pairs and a 5'-terminal phosphate
group that can be chemically modified as described herein (for
example a 5'-terminal phosphate group having Formula IV). In one
embodiment, an asymmetric hairpin siNA molecule of the invention
contains a stem loop motif, wherein the loop portion of the siNA
molecule is biodegradable. In another embodiment, an asymmetric
hairpin siNA molecule of the invention comprises a loop portion
comprising a non-nucleotide linker.
[0281] In another embodiment, a siNA molecule of the invention
comprises an asymmetric double stranded structure having separate
polynucleotide strands comprising sense and antisense regions,
wherein the antisense region is about 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides in length, wherein the sense region is about 3 to about
25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length,
wherein the sense region and the antisense region have at least 3
complementary nucleotides, and wherein the siNA can include one or
more chemical modifications comprising a structure having any of
Formulae I-VII or any combination thereof. For example, an
exemplary chemically-modified siNA molecule of the invention
comprises an asymmetric double stranded structure having separate
polynucleotide strands comprising sense and antisense regions,
wherein the antisense region is about 18 to about 23 (e.g., about
18, 19, 20, 21, 22, or 23) nucleotides in length and wherein the
sense region is about 3 to about 15 (e.g., about 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15) nucleotides in length, wherein the
sense region the antisense region have at least 3 complementary
nucleotides, and wherein the siNA can include one or more chemical
modifications comprising a structure having any of Formulae I-VII
or any combination thereof. In another embodiment, the asymmetric
double stranded siNA molecule can also have a 5'-terminal phosphate
group that can be chemically modified as described herein (for
example a 5'-terminal phosphate group having Formula IV).
[0282] In another embodiment, a siNA molecule of the invention
comprises a circular nucleic acid molecule, wherein the siNA is
about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or
70) nucleotides in length having about 15 to about 30 (e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
base pairs, and wherein the siNA can include a chemical
modification, which comprises a structure having any of Formulae
I-VII or any combination thereof. For example, an exemplary
chemically-modified siNA molecule of the invention comprises a
circular oligonucleotide having about 42 to about 50 (e.g., about
42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that is
chemically-modified with a chemical modification having any of
Formulae I-VII or any combination thereof, wherein the circular
oligonucleotide forms a dumbbell shaped structure having about 19
base pairs and 2 loops.
[0283] In another embodiment, a circular siNA molecule of the
invention contains two loop motifs, wherein one or both loop
portions of the siNA molecule is biodegradable. For example, a
circular siNA molecule of the invention is designed such that
degradation of the loop portions of the siNA molecule in vivo can
generate a double-stranded siNA molecule with 3'-terminal
overhangs, such as 3'-terminal nucleotide overhangs comprising
about 2 nucleotides.
[0284] In one embodiment, a siNA molecule of the invention
comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) abasic moiety, for example a compound having Formula
V:
##STR00056##
wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl,
alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I or
II; R9 is O, S, CH2, S.dbd.O, CHF, or CF2. In one embodiment, R3
and/or R7 comprises a conjugate moiety and a linker (e.g., a
nucleotide or non-nucleotide linker as described herein or
otherwise known in the art). Non-limiting examples of conjugate
moieties include ligands for cellular receptors, such as peptides
derived from naturally occurring protein ligands; protein
localization sequences, including cellular ZIP code sequences;
antibodies; nucleic acid aptamers; vitamins and other co-factors,
such as folate and N-acetylgalactosamine; polymers, such as
polyethyleneglycol (PEG); phospholipids; cholesterol; steroids, and
polyamines, such as PEI, spermine or spermidine.
[0285] In one embodiment, a siNA molecule of the invention
comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) inverted abasic moiety, for example a compound having
Formula VI:
##STR00057##
wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,
F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl,
O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH,
O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl,
alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I or
II; R9 is O, S, CH2, S.dbd.O, CHF, or CF2, and either R2, R3, R8 or
R13 serve as points of attachment to the siNA molecule of the
invention. In one embodiment, R3 and/or R7 comprises a conjugate
moiety and a linker (e.g., a nucleotide or non-nucleotide linker as
described herein or otherwise known in the art). Non-limiting
examples of conjugate moieties include ligands for cellular
receptors, such as peptides derived from naturally occurring
protein ligands; protein localization sequences, including cellular
ZIP code sequences; antibodies; nucleic acid aptamers; vitamins and
other co-factors, such as folate and N-acetylgalactosamine;
polymers, such as polyethyleneglycol (PEG); phospholipids;
cholesterol; steroids, and polyamines, such as PEI, spermine or
spermidine.
[0286] In another embodiment, a siNA molecule of the invention
comprises at least one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) substituted polyalkyl moieties, for example a compound
having Formula VII:
##STR00058##
wherein each n is independently an integer from 1 to 12, each R1,
R2 and R3 is independently H, OH, alkyl, substituted alkyl, alkaryl
or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl,
N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH,
alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH,
alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl,
aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or a group having Formula I,
and R1, R2 or R3 serves as points of attachment to the siNA
molecule of the invention. In one embodiment, R3 and/or R1
comprises a conjugate moiety and a linker (e.g., a nucleotide or
non-nucleotide linker as described herein or otherwise known in the
art). Non-limiting examples of conjugate moieties include ligands
for cellular receptors, such as peptides derived from naturally
occurring protein ligands; protein localization sequences,
including cellular ZIP code sequences; antibodies; nucleic acid
aptamers; vitamins and other co-factors, such as folate and
N-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);
phospholipids; cholesterol; steroids, and polyamines, such as PEI,
spermine or spermidine.
[0287] By "ZIP code" sequences is meant, any peptide or protein
sequence that is involved in cellular topogenic signaling mediated
transport (see for example Ray et al., 2004, Science, 306(1501):
1505)
[0288] In another embodiment, the invention features a compound
having Formula VII, wherein R1 and R2 are hydroxyl (OH) groups,
n=1, and R3 comprises O and is the point of attachment to the
3'-end, the 5'-end, or both of the 3' and 5'-ends of one or both
strands of a double-stranded siNA molecule of the invention or to a
single-stranded siNA molecule of the invention. This modification
is referred to herein as "glyceryl".
[0289] In another embodiment, a chemically modified nucleoside or
non-nucleoside (e.g. a moiety having any of Formula V, VI or VII)
of the invention is at the 3'-end, the 5'-end, or both of the 3'
and 5'-ends of a siNA molecule of the invention. For example,
chemically modified nucleoside or non-nucleoside (e.g., a moiety
having Formula V, VI or VII) can be present at the 3'-end, the
5'-end, or both of the 3' and 5'-ends of the antisense strand, the
sense strand, or both antisense and sense strands of the siNA
molecule. In one embodiment, the chemically modified nucleoside or
non-nucleoside (e.g., a moiety having Formula V, VI or VII) is
present at the 5'-end and 3'-end of the sense strand and the 3'-end
of the antisense strand of a double stranded siNA molecule of the
invention. In one embodiment, the chemically modified nucleoside or
non-nucleoside (e.g., a moiety having Formula V, VI or VII) is
present at the terminal position of the 5'-end and 3'-end of the
sense strand and the 3'-end of the antisense strand of a double
stranded siNA molecule of the invention. In one embodiment, the
chemically modified nucleoside or non-nucleoside (e.g., a moiety
having Formula V, VI or VII) is present at the two terminal
positions of the 5'-end and 3'-end of the sense strand and the
3'-end of the antisense strand of a double stranded siNA molecule
of the invention. In one embodiment, the chemically modified
nucleoside or non-nucleoside (e.g., a moiety having Formula V, VI
or VII) is present at the penultimate position of the 5'-end and
3'-end of the sense strand and the 3'-end of the antisense strand
of a double stranded siNA molecule of the invention. In addition, a
moiety having Formula VII can be present at the 3'-end or the
5'-end of a hairpin siNA molecule as described herein.
[0290] In another embodiment, a siNA molecule of the invention
comprises an abasic residue having Formula V or VI, wherein the
abasic residue having Formula VI or VI is connected to the siNA
construct in a 3'-3', 3'-2', 2'-3', or 5'-5' configuration, such as
at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of one or
both siNA strands.
[0291] In one embodiment, a siNA molecule of the invention
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) locked nucleic acid (LNA) nucleotides, for example, at the
5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination
thereof, of the siNA molecule.
[0292] In one embodiment, a siNA molecule of the invention
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) 4'-thio nucleotides, for example, at the 5'-end, the
3'-end, both of the 5' and 3'-ends, or any combination thereof, of
the siNA molecule.
[0293] In another embodiment, a siNA molecule of the invention
comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more) acyclic nucleotides, for example, at the 5'-end, the
3'-end, both of the 5' and 3'-ends, or any combination thereof, of
the siNA molecule.
[0294] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein any (e.g., one
or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine
nucleotides or alternately a plurality of pyrimidine nucleotides
are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any
(e.g., one or more or all) purine nucleotides present in the sense
region are 2'-deoxy purine nucleotides (e.g., wherein all purine
nucleotides are 2'-deoxy purine nucleotides or alternately a
plurality of purine nucleotides are 2'-deoxy purine
nucleotides).
[0295] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein any (e.g., one
or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-deoxy purine
nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy
purine nucleotides or alternately a plurality of purine nucleotides
are 2'-deoxy purine nucleotides), wherein any nucleotides
comprising a 3'-terminal nucleotide overhang that are present in
said sense region are 2'-deoxy nucleotides.
[0296] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein any (e.g., one
or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-O-methyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides).
[0297] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising a sense region, wherein any (e.g., one
or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), wherein any (e.g., one or more or all)
purine nucleotides present in the sense region are 2'-O-methyl,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides), and wherein any nucleotides comprising a 3'-terminal
nucleotide overhang that are present in said sense region are
2'-deoxy nucleotides.
[0298] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein any (e.g.,
one or more or all) pyrimidine nucleotides present in the antisense
region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-O-methyl,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides).
[0299] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein any (e.g.,
one or more or all) pyrimidine nucleotides present in the antisense
region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), wherein any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-O-methyl,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides), and wherein any nucleotides comprising a 3'-terminal
nucleotide overhang that are present in said antisense region are
2'-deoxy nucleotides.
[0300] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein any (e.g.,
one or more or all) pyrimidine nucleotides present in the antisense
region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-deoxy
purine nucleotides (e.g., wherein all purine nucleotides are
2'-deoxy purine nucleotides or alternately a plurality of purine
nucleotides are 2'-deoxy purine nucleotides).
[0301] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention comprising an antisense region, wherein any (e.g.,
one or more or all) pyrimidine nucleotides present in the antisense
region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and wherein any (e.g., one or more or all)
purine nucleotides present in the antisense region are 2'-O-methyl,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides).
[0302] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid (siNA) molecule
of the invention capable of mediating RNA interference (RNAi)
inside a cell or reconstituted in vitro system comprising a sense
region, wherein one or more pyrimidine nucleotides present in the
sense region are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and one or more purine nucleotides present
in the sense region are 2'-deoxy purine nucleotides (e.g., wherein
all purine nucleotides are 2'-deoxy purine nucleotides or
alternately a plurality of purine nucleotides are 2'-deoxy purine
nucleotides), and an antisense region, wherein one or more
pyrimidine nucleotides present in the antisense region are
2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and one or more purine nucleotides present
in the antisense region are 2'-O-methyl, 4'-thio,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides). The sense region and/or the antisense region can have
a terminal cap modification that is optionally present at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of the sense
and/or antisense sequence. The sense and/or antisense region can
optionally further comprise a 3'-terminal nucleotide overhang
having about 1 to about 4 (e.g., about 1, 2, 3, or 4)
2'-deoxynucleotides. The overhang nucleotides can further comprise
one or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate,
phosphonoacetate, and/or thiophosphonoacetate internucleotide
linkages. In any of these described embodiments, the purine
nucleotides present in the sense region are alternatively
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides or alternately a
plurality of purine nucleotides are 2'-O-methyl, 4'-thio,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides) and one or more
purine nucleotides present in the antisense region are 2'-O-methyl,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides). Also, in any of these embodiments, one or more purine
nucleotides present in the sense region are alternatively purine
ribonucleotides (e.g., wherein all purine nucleotides are purine
ribonucleotides or alternately a plurality of purine nucleotides
are purine ribonucleotides) and any purine nucleotides present in
the antisense region are 2'-O-methyl, 4'-thio,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides). Additionally, in any of these embodiments, one or
more purine nucleotides present in the sense region and/or present
in the antisense region are alternatively selected from the group
consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides,
2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy
nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides and
2'-O-methyl nucleotides (e.g., wherein all purine nucleotides are
selected from the group consisting of 2'-deoxy nucleotides, locked
nucleic acid (LNA) nucleotides, 2'-methoxyethyl nucleotides,
4'-thionucleotides, 2'-O-trifluoromethyl nucleotides,
2'-O-ethyl-trifluoromethoxy nucleotides,
2'-O-difluoromethoxy-ethoxy nucleotides and 2'-O-methyl nucleotides
or alternately a plurality of purine nucleotides are selected from
the group consisting of 2'-deoxy nucleotides, locked nucleic acid
(LNA) nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides,
2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy
nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides and
2'-O-methyl nucleotides).
[0303] In another embodiment, any modified nucleotides present in
the siNA molecules of the invention, preferably in the antisense
strand of the siNA molecules of the invention, but also optionally
in the sense and/or both antisense and sense strands, comprise
modified nucleotides having properties or characteristics similar
to naturally occurring ribonucleotides. For example, the invention
features siNA molecules including modified nucleotides having a
Northern conformation (e.g., Northern pseudorotation cycle, see for
example Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). As such, chemically modified
nucleotides present in the siNA molecules of the invention,
preferably in the antisense strand of the siNA molecules of the
invention, but also optionally in the sense and/or both antisense
and sense strands, are resistant to nuclease degradation while at
the same time maintaining the capacity to mediate RNAi.
Non-limiting examples of nucleotides having a northern
configuration include locked nucleic acid (LNA) nucleotides (e.g.,
2'-O,4'-C-methylene-(D-ribofuranosyl)nucleotides); 2'-methoxyethoxy
(MOE) nucleotides; 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro
nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides,
2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy
nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides, 4'-thio
nucleotides and 2'-O-methyl nucleotides.
[0304] In one embodiment, the sense strand of a double stranded
siNA molecule of the invention comprises a terminal cap moiety,
such as an inverted deoxyabaisc moiety, at the 3'-end, 5'-end, or
both 3' and 5'-ends of the sense strand.
[0305] In one embodiment, the invention features a
chemically-modified short interfering nucleic acid molecule (siNA)
capable of mediating RNA interference (RNAi) inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises a conjugate covalently attached to the
chemically-modified siNA molecule. Non-limiting examples of
conjugates contemplated by the invention include conjugates and
ligands described in Vargeese et al., U.S. Ser. No. 10/427,160,
filed Apr. 30, 2003, incorporated by reference herein in its
entirety, including the drawings. In another embodiment, the
conjugate is covalently attached to the chemically-modified siNA
molecule via a biodegradable linker. In one embodiment, the
conjugate molecule is attached at the 3'-end of either the sense
strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In another embodiment, the
conjugate molecule is attached at the 5'-end of either the sense
strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In yet another embodiment, the
conjugate molecule is attached both the 3'-end and 5'-end of either
the sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule, or any combination thereof. In
one embodiment, a conjugate molecule of the invention comprises a
molecule that facilitates delivery of a chemically-modified siNA
molecule into a biological system, such as a cell. In another
embodiment, the conjugate molecule attached to the
chemically-modified siNA molecule is a ligand for a cellular
receptor, such as peptides derived from naturally occurring protein
ligands; protein localization sequences, including cellular ZIP
code sequences; antibodies; nucleic acid aptamers; vitamins and
other co-factors, such as folate and N-acetylgalactosamine;
polymers, such as polyethyleneglycol (PEG); phospholipids;
cholesterol; steroids, and polyamines, such as PEI, spermine or
spermidine. Examples of specific conjugate molecules contemplated
by the instant invention that can be attached to
chemically-modified siNA molecules are described in Vargeese et
al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002 incorporated by
reference in its entirety herein. The type of conjugates used and
the extent of conjugation of siNA molecules of the invention can be
evaluated for improved pharmacokinetic profiles, bioavailability,
and/or stability of siNA constructs while at the same time
maintaining the ability of the siNA to mediate RNAi activity. As
such, one skilled in the art can screen siNA constructs that are
modified with various conjugates to determine whether the siNA
conjugate complex possesses improved properties while maintaining
the ability to mediate RNAi, for example in animal models as are
generally known in the art.
[0306] In one embodiment, the invention features a short
interfering nucleic acid (siNA) molecule of the invention, wherein
the siNA further comprises a nucleotide, non-nucleotide, or mixed
nucleotide/non-nucleotide linker that joins the sense region of the
siNA to the antisense region of the siNA. In one embodiment, a
nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide
linker is used, for example, to attach a conjugate moiety to the
siNA. In one embodiment, a nucleotide linker of the invention can
be a linker of 2 nucleotides in length, for example about 3, 4, 5,
6, 7, 8, 9, or 10 nucleotides in length. In another embodiment, the
nucleotide linker can be a nucleic acid aptamer.
[0307] In yet another embodiment, a non-nucleotide linker of the
invention comprises abasic nucleotide, polyether, polyamine,
polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, or other
polymeric compounds (e.g. polyethylene glycols such as those having
between 2 and 100 ethylene glycol units). Specific examples include
those described by Seela and Kaiser, Nucleic Acids Res. 1990,
18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz,
J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am.
Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,
21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic
Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &
Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993,
34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al.,
International Publication No. WO 89/02439; Usman et al.,
International Publication No. WO 95/06731; Dudycz et al.,
International Publication No. WO 95/11910 and Ferentz and Verdine,
J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated by
reference herein. A "non-nucleotide" further means any group or
compound that can be incorporated into a nucleic acid chain in the
place of one or more nucleotide units, including either sugar
and/or phosphate substitutions, and allows the remaining bases to
exhibit their enzymatic activity. The group or compound can be
abasic in that it does not contain a commonly recognized nucleotide
base, such as adenosine, guanine, cytosine, uracil or thymine, for
example at the C1 position of the sugar.
[0308] In one embodiment, the invention features a short
interfering nucleic acid (siNA) molecule capable of mediating RNA
interference (RNAi) inside a cell or reconstituted in vitro system,
wherein one or both strands of the siNA molecule that are assembled
from two separate oligonucleotides do not comprise any
ribonucleotides. For example, a siNA molecule can be assembled from
a single oligonculeotide where the sense and antisense regions of
the siNA comprise separate oligonucleotides that do not have any
ribonucleotides (e.g., nucleotides having a 2'-OH group) present in
the oligonucleotides. In another example, a siNA molecule can be
assembled from a single oligonculeotide where the sense and
antisense regions of the siNA are linked or circularized by a
nucleotide or non-nucleotide linker as described herein, wherein
the oligonucleotide does not have any ribonucleotides (e.g.,
nucleotides having a 2'-OH group) present in the oligonucleotide.
Applicant has surprisingly found that the presense of
ribonucleotides (e.g., nucleotides having a 2'-hydroxyl group)
within the siNA molecule is not required or essential to support
RNAi activity. As such, in one embodiment, all positions within the
siNA can include chemically modified nucleotides and/or
non-nucleotides such as nucleotides and or non-nucleotides having
Formula I, II, III, IV, V, VI, or VII or any combination thereof to
the extent that the ability of the siNA molecule to support RNAi
activity in a cell is maintained.
[0309] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system comprising a single stranded
polynucleotide having complementarity to a target nucleic acid
sequence. In another embodiment, the single stranded siNA molecule
of the invention comprises a 5'-terminal phosphate group. In
another embodiment, the single stranded siNA molecule of the
invention comprises a 5'-terminal phosphate group and a 3'-terminal
phosphate group (e.g., a 2',3'-cyclic phosphate). In another
embodiment, the single stranded siNA molecule of the invention
comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet
another embodiment, the single stranded siNA molecule of the
invention comprises one or more chemically modified nucleotides or
non-nucleotides described herein. For example, all the positions
within the siNA molecule can include chemically-modified
nucleotides such as nucleotides having any of Formulae I-VII, or
any combination thereof to the extent that the ability of the siNA
molecule to support RNAi activity in a cell is maintained.
[0310] In one embodiment, a siNA molecule of the invention is a
single stranded siNA molecule that mediates RNAi activity in a cell
or reconstituted in vitro system comprising a single stranded
polynucleotide having complementarity to a target nucleic acid
sequence, wherein one or more pyrimidine nucleotides present in the
siNA are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides
are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy
pyrimidine nucleotides), and wherein any purine nucleotides present
in the antisense region are 2'-O-methyl, 4'-thio,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy, or
2'-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all
purine nucleotides are 2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides or alternately a plurality of purine nucleotides are
2'-O-methyl, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, or 2'-O-difluoromethoxy-ethoxy purine
nucleotides), and a terminal cap modification that is optionally
present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of
the antisense sequence. The siNA optionally further comprises about
1 to about 4 or more (e.g., about 1, 2, 3, 4 or more) terminal
2'-deoxynucleotides at the 3'-end of the siNA molecule, wherein the
terminal nucleotides can further comprise one or more (e.g., 1, 2,
3, 4 or more) phosphorothioate, phosphonoacetate, and/or
thiophosphonoacetate internucleotide linkages, and wherein the siNA
optionally further comprises a terminal phosphate group, such as a
5'-terminal phosphate group. In any of these embodiments, any
purine nucleotides present in the antisense region are
alternatively 2'-deoxy purine nucleotides (e.g., wherein all purine
nucleotides are 2'-deoxy purine nucleotides or alternately a
plurality of purine nucleotides are 2'-deoxy purine nucleotides).
Also, in any of these embodiments, any purine nucleotides present
in the siNA (i.e., purine nucleotides present in the sense and/or
antisense region) can alternatively be locked nucleic acid (LNA)
nucleotides (e.g., wherein all purine nucleotides are LNA
nucleotides or alternately a plurality of purine nucleotides are
LNA nucleotides). Also, in any of these embodiments, any purine
nucleotides present in the siNA are alternatively 2'-methoxyethyl
purine nucleotides (e.g., wherein all purine nucleotides are
2'-methoxyethyl purine nucleotides or alternately a plurality of
purine nucleotides are 2'-methoxyethyl purine nucleotides). In
another embodiment, any modified nucleotides present in the single
stranded siNA molecules of the invention comprise modified
nucleotides having properties or characteristics similar to
naturally occurring ribonucleotides. For example, the invention
features siNA molecules including modified nucleotides having a
Northern conformation (e.g., Northern pseudorotation cycle, see for
example Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). As such, chemically modified
nucleotides present in the single stranded siNA molecules of the
invention are preferably resistant to nuclease degradation while at
the same time maintaining the capacity to mediate RNAi.
[0311] In one embodiment, a siNA molecule of the invention
comprises chemically modified nucleotides or non-nucleotides (e.g.,
having any of Formulae I-VII, such as 2'-deoxy, 2'-deoxy-2'-fluoro,
4'-thio, 2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy or 2'-O-methyl nucleotides) at
alternating positions within one or more strands or regions of the
siNA molecule. For example, such chemical modifications can be
introduced at every other position of a RNA based siNA molecule,
starting at either the first or second nucleotide from the 3'-end
or 5'-end of the siNA. In a non-limiting example, a double stranded
siNA molecule of the invention in which each strand of the siNA is
21 nucleotides in length is featured wherein positions 1, 3, 5, 7,
9, 11, 13, 15, 17, 19 and 21 of each strand are chemically modified
(e.g., with compounds having any of Formulae I-VII, such as such as
2'-deoxy, 2'-deoxy-2'-fluoro, 4'-thio, 2'-O-trifluoromethyl,
2'-O-ethyl-trifluoromethoxy, 2'-O-difluoromethoxy-ethoxy or
2'-O-methyl nucleotides). In another non-limiting example, a double
stranded siNA molecule of the invention in which each strand of the
siNA is 21 nucleotides in length is featured wherein positions 2,
4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are chemically
modified (e.g., with compounds having any of Formulae I-VII, such
as such as 2'-deoxy, 2'-deoxy-2'-fluoro, 4'-thio,
2'-O-trifluoromethyl, 2'-O-ethyl-trifluoromethoxy,
2'-O-difluoromethoxy-ethoxy or 2'-O-methyl nucleotides). Such siNA
molecules can further comprise terminal cap moieties and/or
backbone modifications as described herein.
[0312] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule, such as
a polynucleotide molecule (e.g., siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule) of the invention
to a cell or cells in a subject or organism, comprising
administering a formulated molecular composition of the invention
under conditions suitable for delivery of the polynucleotide
component of the formulated molecular composition to the cell or
cells of the subject or organism. In separate embodiments, the cell
is, for example, a lung cell, liver cell, CNS cell, PNS cell, tumor
cell, kidney cell, vascular cell, skin cell, ocular cell, or cells
of the ear.
[0313] In one embodiment, the invention features a method for
delivering or administering a biologically active molecule, such as
a polynucleotide molecule (e.g., siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule) of the invention
to liver or liver cells (e.g., hepatocytes) in a subject or
organism, comprising administering a formulated molecular
composition of the invention under conditions suitable for delivery
of the polynucleotide component of the formulated molecular
composition to the liver or liver cells (e.g., hepatocytes) of the
subject or organism.
[0314] In one embodiment, the invention features a method for
modulating the expression of a target gene within a cell
comprising, introducing a formulated molecular composition of the
invention into a cell under conditions suitable to modulate the
expression of the target gene in the cell. In one embodiment, the
cell is a liver cell (e.g., hepatocyte). In other embodiments, the
cell is, for example, a lung cell, CNS cell, PNS cell, tumor cell,
kidney cell, vascular cell, skin cell, ocular cell, or cells of the
ear. In one embodiment, the formulated molecular composition
comprises a polynucleotide, such as a siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule.
[0315] In another embodiment, the invention features a method for
modulating the expression of more than one target gene within a
cell comprising, introducing a formulated molecular composition of
the invention into the cell under conditions suitable to modulate
the expression of the target genes in the cell. In one embodiment,
the cell is a liver cell (e.g., hepatocyte). In other embodiments,
the cell is, for example, a lung cell, CNS cell, PNS cell, tumor
cell, kidney cell, vascular cell, skin cell, ocular cell, or cells
of the ear. In one embodiment, the formulated molecular composition
comprises a polynucleotide, such as a siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule.
[0316] In one embodiment, the invention features a method for
treating or preventing a disease, disorder, trait or condition
related to gene expression in a subject or organism comprising
contacting the subject or organism with a formulated molecular
composition of the invention under conditions suitable to modulate
the expression of the target gene in the subject or organism. In
one embodiment, the formulated molecular composition comprises a
polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule. In one embodiment, the reduction of
gene expression and thus reduction in the level of the respective
protein/RNA relieves, to some extent, the symptoms of the disease,
disorder, trait or condition.
[0317] In one embodiment, the invention features a method for
treating or preventing cancer in a subject or organism comprising
contacting the subject or organism with a formulated molecular
composition of the invention under conditions suitable to modulate
the expression of the target gene in the subject or organism
whereby the treatment or prevention of cancer can be achieved. In
one embodiment, the formulated molecular composition comprises a
polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule. In one embodiment, the invention
features contacting the subject or organism with a formulated
molecular composition of the invention via local administration to
relevant tissues or cells, such as cancerous cells and tissues. In
one embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via systemic administration (such as via intravenous or
subcutaneous administration of the formulated molecular
composition) to relevant tissues or cells, such as tissues or cells
involved in the maintenance or development of cancer in a subject
or organism. The formulated molecular composition of the invention
can be formulated or conjugated as described herein or otherwise
known in the art to target appropriate tissues or cells in the
subject or organism.
[0318] In one embodiment, the invention features a method for
treating or preventing a proliferative disease or condition in a
subject or organism comprising contacting the subject or organism
with a formulated molecular composition of the invention under
conditions suitable to modulate the expression of the target gene
in the subject or organism whereby the treatment or prevention of
the proliferative disease or condition can be achieved. In one
embodiment, the formulated molecular composition comprises a
polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule. In one embodiment, the invention
features contacting the subject or organism with a formulated
molecular composition of the invention via local administration to
relevant tissues or cells, such as cells and tissues involved in
proliferative disease. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
proliferative disease or condition in a subject or organism. The
formulated molecular composition of the invention can be formulated
or conjugated as described herein or otherwise known in the art to
target appropriate tissues or cells in the subject or organism.
[0319] In one embodiment, the invention features a method for
treating or preventing transplant and/or tissue rejection
(allograft rejection) in a subject or organism comprising
contacting the subject or organism with a formulated molecular
composition of the invention under conditions suitable to modulate
the expression of the target gene in the subject or organism
whereby the treatment or prevention of transplant and/or tissue
rejection (allograft rejection) can be achieved. In one embodiment,
the formulated molecular composition comprises a polynucleotide,
such as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule. In one embodiment, the invention features contacting
the subject or organism with a formulated molecular composition of
the invention via local administration to relevant tissues or
cells, such as cells and tissues involved in transplant and/or
tissue rejection (allograft rejection). In one embodiment, the
invention features contacting the subject or organism with a
formulated molecular composition of the invention via systemic
administration (such as via intravenous or subcutaneous
administration of the formulated molecular composition) to relevant
tissues or cells, such as tissues or cells involved in the
maintenance or development of transplant and/or tissue rejection
(allograft rejection) in a subject or organism. The formulated
molecular composition of the invention can be formulated or
conjugated as described herein or otherwise known in the art to
target appropriate tissues or cells in the subject or organism.
[0320] In one embodiment, the invention features a method for
treating or preventing an autoimmune disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the autoimmune disease, disorder, trait or condition
can be achieved. In one embodiment, the formulated molecular
composition comprises a polynucleotide, such as a siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule. In one
embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the autoimmune disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
autoimmune disease, disorder, trait or condition in a subject or
organism. The formulated molecular composition of the invention can
be formulated or conjugated as described herein or otherwise known
in the art to target appropriate tissues or cells in the subject or
organism.
[0321] In one embodiment, the invention features a method for
treating or preventing an infectious disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the infectious disease, disorder, trait or condition
can be achieved. In one embodiment, the formulated molecular
composition comprises a polynucleotide, such as a siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule. In one
embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the infectious disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
infectious disease, disorder, trait or condition in a subject or
organism. The formulated molecular composition of the invention can
be formulated or conjugated as described herein or otherwise known
in the art to target appropriate tissues or cells in the subject or
organism.
[0322] In one embodiment, the invention features a method for
treating or preventing an age-related disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the age-related disease, disorder, trait or condition
can be achieved. In one embodiment, the formulated molecular
composition comprises a polynucleotide, such as a siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule. In one
embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the age-related disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
age-related disease, disorder, trait or condition in a subject or
organism. The formulated molecular composition of the invention can
be formulated or conjugated as described herein or otherwise known
in the art to target appropriate tissues or cells in the subject or
organism.
[0323] In one embodiment, the invention features a method for
treating or preventing a neurologic or neurodegenerative disease,
disorder, trait or condition in a subject or organism comprising
contacting the subject or organism with a formulated molecular
composition of the invention under conditions suitable to modulate
the expression of the target gene in the subject or organism
whereby the treatment or prevention of the neurologic or
neurodegenerative disease, disorder, trait or condition can be
achieved. In one embodiment, the formulated molecular composition
comprises a polynucleotide, such as a siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule. In one embodiment,
the invention features contacting the subject or organism with a
formulated molecular composition of the invention via local
administration to relevant tissues or cells, such as cells and
tissues involved in the neurologic or neurodegenerative disease,
disorder, trait or condition. In one embodiment, the invention
features contacting the subject or organism with a formulated
molecular composition of the invention via systemic administration
(such as via catheterization, osmotic pump administration (e.g.,
intrathecal or ventricular) intravenous or subcutaneous
administration of the formulated molecular composition) to relevant
tissues or cells, such as tissues or cells involved in the
maintenance or development of the neurologic or neurodegenerative
disease, disorder, trait or condition in a subject or organism. The
formulated molecular composition of the invention can be formulated
or conjugated as described herein or otherwise known in the art to
target appropriate tissues or cells in the subject or organism. In
one embodiment, the neurologic disease is Huntington disease.
[0324] In one embodiment, the invention features a method for
treating or preventing a metabolic disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the metabolic disease, disorder, trait or condition
can be achieved. In one embodiment, the formulated molecular
composition comprises a polynucleotide, such as a siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule. In one
embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the metabolic disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
metabolic disease, disorder, trait or condition in a subject or
organism. The formulated molecular composition of the invention can
be formulated or conjugated as described herein or otherwise known
in the art to target appropriate tissues or cells in the subject or
organism.
[0325] In one embodiment, the invention features a method for
treating or preventing a cardiovascular disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the cardiovascular disease, disorder, trait or
condition can be achieved. In one embodiment, the formulated
molecular composition comprises a polynucleotide, such as a siNA,
miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,
triplex forming oligonucleotide, or other nucleic acid molecule. In
one embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the cardiovascular disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
cardiovascular disease, disorder, trait or condition in a subject
or organism. The formulated molecular composition of the invention
can be formulated or conjugated as described herein or otherwise
known in the art to target appropriate tissues or cells in the
subject or organism.
[0326] In one embodiment, the invention features a method for
treating or preventing a respiratory disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the respiratory disease, disorder, trait or condition
can be achieved. In one embodiment, the formulated molecular
composition comprises a polynucleotide, such as a siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule. In one
embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the respiratory disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
respiratory disease, disorder, trait or condition in a subject or
organism. The formulated molecular composition of the invention can
be formulated or conjugated as described herein or otherwise known
in the art to target appropriate tissues or cells in the subject or
organism.
[0327] In one embodiment, the invention features a method for
treating or preventing an ocular disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the ocular disease, disorder, trait or condition can
be achieved. In one embodiment, the formulated molecular
composition comprises a polynucleotide, such as a siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule. In one
embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the ocular disease, disorder, trait
or condition. In one embodiment, the invention features contacting
the subject or organism with a formulated molecular composition of
the invention via systemic administration (such as via intravenous
or subcutaneous administration of the formulated molecular
composition) to relevant tissues or cells, such as tissues or cells
involved in the maintenance or development of the ocular disease,
disorder, trait or condition in a subject or organism. The
formulated molecular composition of the invention can be formulated
or conjugated as described herein or otherwise known in the art to
target appropriate tissues or cells in the subject or organism.
[0328] In one embodiment, the invention features a method for
treating or preventing a dermatological disease, disorder, trait or
condition in a subject or organism comprising contacting the
subject or organism with a formulated molecular composition of the
invention under conditions suitable to modulate the expression of
the target gene in the subject or organism whereby the treatment or
prevention of the dermatological disease, disorder, trait or
condition can be achieved. In one embodiment, the formulated
molecular composition comprises a polynucleotide, such as a siNA,
miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,
triplex forming oligonucleotide, or other nucleic acid molecule. In
one embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via local administration to relevant tissues or cells, such as
cells and tissues involved in the dermatological disease, disorder,
trait or condition. In one embodiment, the invention features
contacting the subject or organism with a formulated molecular
composition of the invention via systemic administration (such as
via intravenous or subcutaneous administration of the formulated
molecular composition) to relevant tissues or cells, such as
tissues or cells involved in the maintenance or development of the
dermatological disease, disorder, trait or condition in a subject
or organism. The formulated molecular composition of the invention
can be formulated or conjugated as described herein or otherwise
known in the art to target appropriate tissues or cells in the
subject or organism.
[0329] In one embodiment, the invention features a method for
treating or preventing a liver disease, disorder, trait or
condition (e.g., hepatitis, HCV, HBV, diabetes, cirrhosis,
hepatocellular carcinoma etc.) in a subject or organism comprising
contacting the subject or organism with a formulated molecular
composition of the invention under conditions suitable to modulate
the expression of the target gene in the subject or organism
whereby the treatment or prevention of the liver disease, disorder,
trait or condition can be achieved. In one embodiment, the
formulated molecular composition comprises a polynucleotide, such
as a siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule. In one embodiment, the invention features contacting
the subject or organism with a formulated molecular composition of
the invention via local administration to relevant tissues or
cells, such as liver cells and tissues involved in the liver
disease, disorder, trait or condition. In one embodiment, the
invention features contacting the subject or organism with a
formulated molecular composition of the invention via systemic
administration (such as via intravenous or subcutaneous
administration of the formulated molecular composition) to relevant
tissues or cells, such as tissues or cells involved in the
maintenance or development of the liver disease, disorder, trait or
condition in a subject or organism. The formulated molecular
composition of the invention can be formulated or conjugated as
described herein or otherwise known in the art to target
appropriate tissues or cells in the subject or organism.
[0330] In one embodiment, the invention features a method for
treating or preventing a kidney/renal disease, disorder, trait or
condition (e.g., polycystic kidney disease etc.) in a subject or
organism comprising contacting the subject or organism with a
formulated molecular composition of the invention under conditions
suitable to modulate the expression of the target gene in the
subject or organism whereby the treatment or prevention of the
kidney/renal disease, disorder, trait or condition can be achieved.
In one embodiment, the formulated molecular composition comprises a
polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule. In one embodiment, the invention
features contacting the subject or organism with a formulated
molecular composition of the invention via local administration to
relevant tissues or cells, such as kidney/renal cells and tissues
involved in the kidney/renal disease, disorder, trait or condition.
In one embodiment, the invention features contacting the subject or
organism with a formulated molecular composition of the invention
via systemic administration (such as via intravenous or
subcutaneous administration of the formulated molecular
composition) to relevant tissues or cells, such as tissues or cells
involved in the maintenance or development of the kidney/renal
disease, disorder, trait or condition in a subject or organism. The
formulated molecular composition of the invention can be formulated
or conjugated as described herein or otherwise known in the art to
target appropriate tissues or cells in the subject or organism.
[0331] In one embodiment, the invention features a method for
treating or preventing an auditory disease, disorder, trait or
condition (e.g., hearing loss, deafness, etc.) in a subject or
organism comprising contacting the subject or organism with a
formulated molecular composition of the invention under conditions
suitable to modulate the expression of the target gene in the
subject or organism whereby the treatment or prevention of the
auditory disease, disorder, trait or condition can be achieved. In
one embodiment, the formulated molecular composition comprises a
polynucleotide, such as a siNA, miRNA, RNAi inhibitor, antisense,
aptamer, decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or
other nucleic acid molecule. In one embodiment, the invention
features contacting the subject or organism with a formulated
molecular composition of the invention via local administration to
relevant tissues or cells, such as cells and tissues of the ear,
inner hear, or middle ear involved in the auditory disease,
disorder, trait or condition. In one embodiment, the invention
features contacting the subject or organism with a formulated
molecular composition of the invention via systemic administration
(such as via intravenous or subcutaneous administration of the
formulated molecular composition) to relevant tissues or cells,
such as tissues or cells involved in the maintenance or development
of the auditory disease, disorder, trait or condition in a subject
or organism. The formulated molecular composition of the invention
can be formulated or conjugated as described herein or otherwise
known in the art to target appropriate tissues or cells in the
subject or organism.
[0332] In one embodiment, the invention features a method for
treating or preventing a disease or condition as described herein
in a subject or organism, comprising administering to the subject
or organism a formulated molecular composition of the invention;
wherein the formulated molecular composition is administered under
conditions suitable for reducing or inhibiting the level of target
gene expression in the subject compared to a subject not treated
with the formulated molecular composition. In one embodiment, the
formulated molecular composition comprises a lipid nanoparticle and
a siNA molecule of the invention.
[0333] In one embodiment, the invention features a method for
treating or preventing a disease or condition as described herein
in a subject or organism, comprising administering to the subject a
formulated molecular composition of the invention; wherein (a) the
formulated molecular composition comprises a double stranded
nucleic acid molecule having a sense strand and an antisense
strand; (b) each strand of the double stranded nucleic acid
molecule is 15 to 28 nucleotides in length; (c) at least 15
nucleotides of the sense strand are complementary to the antisense
strand (d) the antisense strand of the double stranded nucleic acid
molecule has complementarity to a target RNA; and wherein the
formulated molecular composition is administered under conditions
suitable for reducing or inhibiting the target RNA in the subject
compared to a subject not treated with the formulated molecular
composition. In one embodiment, the formulated molecular
composition comprises a lipid nanoparticle and a siNA molecule of
the invention.
[0334] In one embodiment, the invention features a method for
treating or preventing a disease or condition as described herein
in a subject or organism, comprising administering to the subject a
formulated molecular composition of the invention; wherein (a) the
formulated molecular composition comprises a double stranded
nucleic acid molecule having a sense strand and an antisense
strand; (b) each strand of the double stranded nucleic acid
molecule is 15 to 28 nucleotides in length; (c) at least 15
nucleotides of the sense strand are complementary to the antisense
strand (d) the antisense strand of the double stranded nucleic acid
molecule has complementarity to a target RNA; (e) at least 20% of
the internal nucleotides of each strand of the double stranded
nucleic acid molecule are modified nucleosides having a chemical
modification; and (f) at least two of the chemical modifications
are different from each other, and wherein the formulated molecular
composition is administered under conditions suitable for reducing
or inhibiting the level of target RNA in the subject compared to a
subject not treated with the formulated molecular composition. In
one embodiment, the formulated molecular composition comprises a
lipid nanoparticle and a siNA molecule of the invention.
[0335] In any of the methods of treatment of the invention, the
formulated molecular composition can be administered to the subject
as a course of treatment, for example administration at various
time intervals, such as once per day over the course of treatment,
once every two days over the course of treatment, once every three
days over the course of treatment, once every four days over the
course of treatment, once every five days over the course of
treatment, once every six days over the course of treatment, once
per week over the course of treatment, once every other week over
the course of treatment, once per month over the course of
treatment, etc. In one embodiment, the course of treatment is once
every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In one embodiment,
the course of treatment is from about one to about 52 weeks or
longer (e.g., indefinitely). In one embodiment, the course of
treatment is from about one to about 48 months or longer (e.g.,
indefinitely).
[0336] In one embodiment, a course of treatment involves an initial
course of treatment, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more weeks for a fixed interval (e.g., 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., 10.times. or more) followed by a maintenance course of
treatment, such as once every 4, 6, 8, 10, 15, 20, 25, 30, 35, 40,
or more weeks for an additional fixed interval (e.g., 1.times.,
2.times., 3.times., 4.times., 5.times., 6.times., 7.times.,
8.times., 9.times., 10.times. or more).
[0337] In any of the methods of treatment of the invention, the
formulated molecular composition can be administered to the subject
systemically as described herein or otherwise known in the art.
Systemic administration can include, for example, intravenous,
subcutaneous, intramuscular, catheterization, nasopharangeal,
transdermal, or gastrointestinal administration as is generally
known in the art.
[0338] In one embodiment, in any of the methods of treatment or
prevention of the invention, the formulated molecular composition
can be administered to the subject locally or to local tissues as
described herein or otherwise known in the art. Local
administration can include, for example, catheterization,
implantation, osmotic pumping, direct injection, dermal/transdermal
application, stenting, ear/eye drops, or portal vein administration
to relevant tissues, or any other local administration technique,
method or procedure, as is generally known in the art.
[0339] In one embodiment, the invention features a composition
comprising a formulated molecular composition of the invention, in
a pharmaceutically acceptable carrier or diluent. In another
embodiment, the invention features a pharmaceutical composition
comprising formulated molecular compositions of the invention,
targeting one or more genes in a pharmaceutically acceptable
carrier or diluent. In another embodiment, the invention features a
method for diagnosing a disease or condition in a subject
comprising administering to the subject a formulated molecular
composition of the invention under conditions suitable for the
diagnosis of the disease or condition in the subject. In another
embodiment, the invention features a method for treating or
preventing a disease, trait, or condition in a subject, comprising
administering to the subject a formulated molecular composition of
the invention under conditions suitable for the treatment or
prevention of the disease, trait or condition in the subject, alone
or in conjunction with one or more other therapeutic compounds.
[0340] In one embodiment, the method of synthesis of polynucleotide
molecules of the invention, including but not limited to siNA,
miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,
triplex forming oligonucleotide, or other nucleic acid molecules,
comprises the teachings of Scaringe et al., U.S. Pat. Nos.
5,889,136; 6,008,400; and 6,111,086, incorporated by reference
herein in their entirety.
[0341] In another embodiment, the invention features a method for
generating formulated polynucleotide (e.g., to siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule)
compositions with increased nuclease resistance comprising (a)
introducing modified nucleotides into a polynucleotide component of
a formulated molecular composition of the invention, and (b)
assaying the formulated molecular composition of step (a) under
conditions suitable for isolating formulated polynucleotide
compositions having increased nuclease resistance.
[0342] In another embodiment, the invention features a method for
generating polynucleotide (e.g., to siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule) molecules with
improved toxicologic profiles (e.g., having attenuated or no
immunstimulatory properties) comprising (a) introducing nucleotides
having any of Formula I-VII (e.g., siNA motifs referred to in Table
I) or any combination thereof into a polynucleotide molecule, and
(b) assaying the polynucleotide molecule of step (a) under
conditions suitable for isolating siNA molecules having improved
toxicologic profiles.
[0343] In another embodiment, the invention features a method for
generating formulated siNA compositions with improved toxicologic
profiles (e.g., having attenuated or no immunstimulatory
properties) comprising (a) generating a formulated siNA composition
comprising a siNA molecule of the invention and a delivery vehicle
or delivery particle as described herein or as otherwise known in
the art, and (b) assaying the siNA formualtion of step (a) under
conditions suitable for isolating formulated siNA compositions
having improved toxicologic profiles.
[0344] In another embodiment, the invention features a method for
generating siNA molecules that do not stimulate an interferon
response (e.g., no interferon response or attenuated interferon
response) in a cell, subject, or organism, comprising (a)
introducing nucleotides having any of Formula I-VII (e.g., siNA
motifs referred to in Table I) or any combination thereof into a
siNA molecule, and (b) assaying the siNA molecule of step (a) under
conditions suitable for isolating siNA molecules that do not
stimulate an interferon response.
[0345] In another embodiment, the invention features a method for
generating formulated siNA compositions that do not stimulate an
interferon response (e.g., no interferon response or attenuated
interferon response) in a cell, subject, or organism, comprising
(a) generating a formulated siNA composition comprising a siNA
molecule of the invention and a delivery vehicle or delivery
particle as described herein or as otherwise known in the art, and
(b) assaying the siNA formualtion of step (a) under conditions
suitable for isolating formulated siNA compositions that do not
stimulate an interferon response. In one embodiment, the interferon
comprises interferon alpha.
[0346] In another embodiment, the invention features a method for
generating siNA molecules that do not stimulate an inflammatory or
proinflammatory cytokine response (e.g., no cytokine response or
attenuated cytokine response) in a cell, subject, or organism,
comprising (a) introducing nucleotides having any of Formula I-VII
(e.g., siNA motifs referred to in Table I) or any combination
thereof into a siNA molecule, and (b) assaying the siNA molecule of
step (a) under conditions suitable for isolating siNA molecules
that do not stimulate a cytokine response. In one embodiment, the
cytokine comprises an interleukin such as interleukin-6 (IL-6)
and/or tumor necrosis factor alpha (TNF-.alpha.).
[0347] In another embodiment, the invention features a method for
generating formulated siNA compositions that do not stimulate an
inflammatory or proinflammatory cytokine response (e.g., no
cytokine response or attenuated cytokine response) in a cell,
subject, or organism, comprising (a) generating a formulated siNA
composition comprising a siNA molecule of the invention and a
delivery vehicle or delivery particle as described herein or as
otherwise known in the art, and (b) assaying the siNA formualtion
of step (a) under conditions suitable for isolating formulated siNA
compositions that do not stimulate a cytokine response. In one
embodiment, the cytokine comprises an interleukin such as
interleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-.alpha.).
[0348] In another embodiment, the invention features a method for
generating siNA molecules that do not stimulate Toll-like Receptor
(TLR) response (e.g., no TLR response or attenuated TLR response)
in a cell, subject, or organism, comprising (a) introducing
nucleotides having any of Formula I-VII (e.g., siNA motifs referred
to in Table I) or any combination thereof into a siNA molecule, and
(b) assaying the siNA molecule of step (a) under conditions
suitable for isolating siNA molecules that do not stimulate a TLR
response. In one embodiment, the TLR comprises TLR3, TLR7, TLR8
and/or TLR9.
[0349] In another embodiment, the invention features a method for
generating formulated siNA compositions that do not stimulate a
Toll-like Receptor (TLR) response (e.g., no TLR response or
attenuated TLR response) in a cell, subject, or organism,
comprising (a) generating a formulated siNA composition comprising
a siNA molecule of the invention and a delivery vehicle or delivery
particle as described herein or as otherwise known in the art, and
(b) assaying the siNA formualtion of step (a) under conditions
suitable for isolating formulated siNA compositions that do not
stimulate a TLR response. In one embodiment, the TLR comprises
TLR3, TLR7, TLR8 and/or TLR9.
[0350] By "improved toxicologic profile", is meant that the
polynucleotide, formulated molecular composition, siNA or
formulated siNA composition exhibits decreased toxicity in a cell,
subject, or organism compared to an unmodified polynucleotide,
formulated molecular composition, siNA or formulated siNA
composition, or siNA molecule having fewer modifications or
modifications that are less effective in imparting improved
toxicology. In a non-limiting example, polynucleotides, formulated
molecular compositions, siNAs or formulated siNA compositions with
improved toxicologic profiles are associated with reduced
immunostimulatory properties, such as a reduced, decreased or
attenuated immunostimulatory response in a cell, subject, or
organism compared to an unmodified polynucleotide, formulated
molecular composition, siNA or formulated siNA composition, or
polynucleotide (e.g., siNA) molecule having fewer modifications or
modifications that are less effective in imparting improved
toxicology. Such an improved toxicologic profile is characterized
by abrogated or reduced immunostimulation, such as reduction or
abrogation of induction of interferons (e.g., interferon alpha),
inflammatory cytokines (e.g., interleukins such as IL-6, and/or
TNF-alpha), and/or toll like receptors (e.g., TLR-3, TLR-7, TLR-8,
and/or TLR-9). In one embodiment, a polynucleotide, formulated
molecular composition, siNA or formulated siNA composition with an
improved toxicological profile comprises no ribonucleotides. In one
embodiment, a polynucleotide, formulated molecular composition,
siNA or formulated siNA composition with an improved toxicological
profile comprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4
ribonucleotides). In one embodiment, a siNA or formulated siNA
composition with an improved toxicological profile comprises Stab
7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18,
Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab
28, Stab 29, Stab 30, Stab 31, Stab 32, Stab 33, Stab 34 or any
combination thereof (see Table I). Herein, numeric Stab chemistries
include both 2'-fluoro and 2'-OCF3 versions of the chemistries
shown in Table IV. For example, "Stab 7/8" refers to both Stab 7/8
and Stab 7F/8F etc. In one embodiment, a siNA or formulated siNA
composition with an improved toxicological profile comprises a siNA
molecule as described in United States Patent Application
Publication No. 20030077829, incorporated by reference herein in
its entirety including the drawings.
[0351] In one embodiment, the level of immunostimulatory response
associated with a given polynucleotide, formulated molecular
composition, siNA molecule or formulated siNA composition can be
measured as is described herein or as is otherwise known in the
art, for example by determining the level of PKR/interferon
response, proliferation, B-cell activation, and/or cytokine
production in assays to quantitate the immunostimulatory response
of particular polynucleotide molecules (see, for example, Leifer et
al., 2003, J Immunother. 26, 313-9; and U.S. Pat. No. 5,968,909,
incorporated in its entirety by reference). In one embodiment, the
reduced immunostimulatory response is between about 10% and about
100% compared to an unmodified or minimally modified siRNA
molecule, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or 100% reduced immunostimulatory response. In one embodiment, the
immunostimulatory response associated with a siNA molecule can be
modulated by the degree of chemical modification. For example, a
siNA molecule having between about 10% and about 100%, e.g., about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the
nucleotide positions in the siNA molecule modified can be selected
to have a corresponding degree of immunostimulatory properties as
described herein.
[0352] In one embodiment, the degree of reduced immunostimulatory
response is selected for optimized RNAi activity. For example,
retaining a certain degree of immunostimulation can be preferred to
treat viral infection, where less than 100% reduction in
immunostimulation may be preferred for maximal antiviral activity
(e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
reduction in immunostimulation) whereas the inhibition of
expression of an endogenous gene target may be preferred with siNA
molecules that posess minimal immunostimulatory properties to
prevent non-specific toxicity or off target effects (e.g., about
90% to about 100% reduction in immunostimulation).
[0353] In one embodiment, a formulated siNA composition of the
invention is designed such that the composition is not toxic to
cells or has a minimized toxicicological profile such that the
composition does not interfere with the efficacy of RNAi mediated
by the siNA component of the formulated siNA composition or result
in toxicity to the cells.
[0354] The term "formulated molecular composition" or "lipid
nanoparticle", or "lipid nanoparticle composition" or "LNP as used
herein refers to a composition comprising one or more biologically
active molecules independently or in combination with a cationic
lipid, a neutral lipid, and/or a polyethyleneglycol-diacylglycerol
(i.e., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB) conjugate. A formulated molecular
composition can further comprise cholesterol or a cholesterol
derivative (see FIG. 5). The cationic lipid of the invention can
comprise a compound having any of Formulae CLI, CLII, CLIII, CLIV,
CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII, CLXIV,
CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII,
CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI,
CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII,
CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),
1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),
1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),
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-dimethy-1-(cis,cis-9',12-
'-octadecadienoxy)propane (CpLin DMA),
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture
thereof. The neutral lipid can comprise
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. The PEG conjugate can comprise a
PEG-dilaurylglycerol (C12), a PEG-dimyristylglycerol (C14), a
PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18),
PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14),
PEG-dipalmitoylglycamide (C16), PEG-disterylglycamide (C18),
PEG-cholesterol, or PEG-DMB. The cationic lipid component can
comprise from about 2% to about 60%, from about 5% to about 45%,
from about 5% to about 15%, or from about 40% to about 50% of the
total lipid present in the formulation. The neutral lipid component
can comprise from about 5% to about 90%, or from about 20% to about
85% of the total lipid present in the formulation. The PEG-DAG
conjugate (e.g., polyethyleneglycol diacylglycerol (PEG-DAG),
PEG-cholesterol, or PEG-DMB) can comprise from about 1% to about
20%, or from about 4% to about 15% of the total lipid present in
the formulation. The cholesterol component can comprise from about
10% to about 60%, or from about 20% to about 45% of the total lipid
present in the formulation. In one embodiment, a formulated
molecular composition of the invention comprises a cationic lipid
component comprising about 7.5% of the total lipid present in the
formulation, a neutral lipid comprising about 82.5% of the total
lipid present in the formulation, and a PEG conjugate comprising
about 10% of the total lipid present in the formulation. In one
embodiment, a formulated molecular composition of the invention
comprises a biologically active molecule, DODMA, DSPC, and a
PEG-DAG conjugate. In one embodiment, the PEG-DAG conjugate is
PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),
PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In
another embodiment, the formulated molecular composition also
comprises cholesterol or a cholesterol derivative. In one
embodiment, the formulated molecular composition comprises a lipid
nanoparticle formulation as shown in Table IV.
[0355] The term "formulated siNA composition" as used herein refers
to a composition comprising one or more siNA molecules or a vector
encoding one or more siNA molecules independently or in combination
with a cationic lipid, a neutral lipid, and/or a
polyethyleneglycol-diacylglycerol (PEG-DAG) or PEG-cholesterol
(PEG-Chol) conjugate. A formulated siNA composition can further
comprise cholesterol or a cholesterol derivative. The cationic
lipid of the invention can comprise a compound having any of
Formulae CLI, CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX,
CLX, CLXI, CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVII, CLXVIII,
CLXIX, CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV, CLXXVI,
CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI, CLXXXII, CLXXXIII,
CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII, CLXXXIX, CLXXXX,
CLXXXXI, CLXXXXII, N,N-dioleyl-N,N-dimethylammonium chloride
(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),
1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),
1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),
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-dimethy-1-(cis,cis-9',12-
'-octadecadienoxy)propane (CpLin DMA),
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture
thereof. The neutral lipid can comprise a compound having any of
Formulae NLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. The PEG conjugate can comprise a
PEG-dilaurylglycerol (C12), a PEG-dimyristylglycerol (C14), a
PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18),
PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14),
PEG-dipalmitoylglycamide (C16), PEG-disterylglycamide (C18),
PEG-cholesterol, or PEG-DMB. The cationic lipid component can
comprise from about 2% to about 60%, from about 5% to about 45%,
from about 5% to about 15%, or from about 40% to about 50% of the
total lipid present in the formulation. The neutral lipid component
can comprise from about 5% to about 90%, or from about 20% to about
85% of the total lipid present in the formulation. The PEG-DAG
conjugate can comprise from about 1% to about 20%, or from about 4%
to about 15% of the total lipid present in the formulation. The
cholesterol component can comprise from about 10% to about 60%, or
from about 20% to about 45% of the total lipid present in the
formulation. In one embodiment, a formulated siNA composition of
the invention comprises a cationic lipid component comprising about
7.5% of the total lipid present in the formulation, a neutral lipid
comprising about 82.5% of the total lipid present in the
formulation, and a PEG-DAG conjugate comprising about 10% of the
total lipid present in the formulation. In one embodiment, a
formulated siNA composition of the invention comprises a siNA
molecule, DODMA, DSPC, and a PEG-DAG conjugate. In one embodiment,
the PEG-DAG conjugate is PEG-dilaurylglycerol (C12),
PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), or
PEG-disterylglycerol (C18). In another embodiment, the formulated
siNA composition also comprises cholesterol or a cholesterol
derivative.
[0356] The term "formulated miRNA composition" as used herein
refers to a composition comprising one or more miRNA molecules or a
vector encoding one or more miRNA molecules independently or in
combination with a cationic lipid, a neutral lipid, and/or a
polyethyleneglycol-diacylglycerol (PEG-DAG) or PEG-cholesterol
(PEG-Chol) conjugate. A formulated miRNA composition can further
comprise cholesterol or a cholesterol derivative. The cationic
lipid of the invention can comprise a compound having any of
Formulae CLI, CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX,
CLX, CLXI, CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVII, CLXVIII,
CLXIX, CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV, CLXXVI,
CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI, CLXXXII, CLXXXIII,
CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII, CLXXXIX, CLXXXX,
CLXXXXI, CLXXXXII, N,N-dioleyl-N,N-dimethylammonium chloride
(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),
1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),
1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),
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-dimethy-1-(cis,cis-9',12-
'-octadecadienoxy)propane (CpLin DMA),
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture
thereof. The neutral lipid can comprise a compound having any of
Formulae NLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. The PEG conjugate can comprise a
PEG-dilaurylglycerol (C12), a PEG-dimyristylglycerol (C14), a
PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18),
PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14),
PEG-dipalmitoylglycamide (C16), PEG-disterylglycamide (C18),
PEG-cholesterol, or PEG-DMB. The cationic lipid component can
comprise from about 2% to about 60%, from about 5% to about 45%,
from about 5% to about 15%, or from about 40% to about 50% of the
total lipid present in the formulation. The neutral lipid component
can comprise from about 5% to about 90%, or from about 20% to about
85% of the total lipid present in the formulation. The PEG-DAG
conjugate can comprise from about 1% to about 20%, or from about 4%
to about 15% of the total lipid present in the formulation. The
cholesterol component can comprise from about 10% to about 60%, or
from about 20% to about 45% of the total lipid present in the
formulation. In one embodiment, a formulated miRNA composition of
the invention comprises a cationic lipid component comprising about
7.5% of the total lipid present in the formulation, a neutral lipid
comprising about 82.5% of the total lipid present in the
formulation, and a PEG-DAG conjugate comprising about 10% of the
total lipid present in the formulation. In one embodiment, a
formulated miRNA composition of the invention comprises a miRNA
molecule, DODMA, DSPC, and a PEG-DAG conjugate. In one embodiment,
the PEG-DAG conjugate is PEG-dilaurylglycerol (C12),
PEG-dimyristylglycerol (C14), PEG-dipalmitoylglycerol (C16), or
PEG-disterylglycerol (C18). In another embodiment, the formulated
miRNA composition also comprises cholesterol or a cholesterol
derivative.
[0357] The term "formulated RNAi inhibitor composition" as used
herein refers to a composition comprising one or more RNAi
inhibitor molecules or a vector encoding one or more RNAi inhibitor
molecules independently or in combination with a cationic lipid, a
neutral lipid, and/or a polyethyleneglycol-diacylglycerol (PEG-DAG)
or PEG-cholesterol (PEG-Chol) conjugate. A formulated RNAi
inhibitor composition can further comprise cholesterol or a
cholesterol derivative. The cationic lipid of the invention can
comprise a compound having any of Formulae CLI, CLII, CLIII, CLIV,
CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI, CLXII, CLXIII, CLXIV,
CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII,
CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI,
CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII,
CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP),
1,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP),
1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP),
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-dimethy-1-(cis,cis-9',12-
'-octadecadienoxy)propane (CpLin DMA),
N,N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and/or a mixture
thereof. The neutral lipid can comprise a compound having any of
Formulae NLI-NLVII, dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and/or a
mixture thereof. The PEG conjugate can comprise a
PEG-dilaurylglycerol (C12), a PEG-dimyristylglycerol (C14), a
PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18),
PEG-dilaurylglycamide (C12), PEG-dimyristylglycamide (C14),
PEG-dipalmitoylglycamide (C16), PEG-disterylglycamide (C18),
PEG-cholesterol, or PEG-DMB. The cationic lipid component can
comprise from about 2% to about 60%, from about 5% to about 45%,
from about 5% to about 15%, or from about 40% to about 50% of the
total lipid present in the formulation. The neutral lipid component
can comprise from about 5% to about 90%, or from about 20% to about
85% of the total lipid present in the formulation. The PEG-DAG
conjugate can comprise from about 1% to about 20%, or from about 4%
to about 15% of the total lipid present in the formulation. The
cholesterol component can comprise from about 10% to about 60%, or
from about 20% to about 45% of the total lipid present in the
formulation. In one embodiment, a formulated RNAi inhibitor
composition of the invention comprises a cationic lipid component
comprising about 7.5% of the total lipid present in the
formulation, a neutral lipid comprising about 82.5% of the total
lipid present in the formulation, and a PEG-DAG conjugate
comprising about 10% of the total lipid present in the formulation.
In one embodiment, a formulated RNAi inhibitor composition of the
invention comprises a RNAi inhibitor molecule, DODMA, DSPC, and a
PEG-DAG conjugate. In one embodiment, the PEG-DAG conjugate is
PEG-dilaurylglycerol (C12), PEG-dimyristylglycerol (C14),
PEG-dipalmitoylglycerol (C16), or PEG-disterylglycerol (C18). In
another embodiment, the formulated RNAi inhibitor composition also
comprises cholesterol or a cholesterol derivative.
[0358] By "cationic lipid" as used herein is meant any lipophilic
compound having cationic change, such as a compound having any of
Formulae CLI-CLXXXXII.
[0359] By "neutral lipid" as used herein is meant any lipophilic
compound having non-cationic change (e.g., anionic or neutral
charge).
[0360] By "PEG" is meant, any polyethylene glycol or other
polyalkylene ether or equivalent polymer. In one embodiment, the
PEG is a PEG conjugate which can comprise a 200 to 10,000 atom PEG
molecule linked to, or example, a lipid moiety of the invention. In
one embodiment, the PEG is a polydispersion represented by the
formula PEG.sub.n, where n=about 33 to 67 for a 1500 Da to 3000 Da
PEG, average=45 for 2KPEG/PEG2000.
[0361] By "nanoparticle" is meant a microscopic particle whose size
is measured in nanometers. Nanoparticles of the invention typically
range from about 1 to about 999 nm in diameter, and can include an
encapsulated or enclosed biologically active molecule.
[0362] By "microparticle" is meant a is a microscopic particle
whose size is measured in micrometers. Microparticles of the
invention typically range from about 1 to about 100 micrometers in
diameter, and can include an encapsulated or enclosed biologically
active molecule.
[0363] The terms "short interfering nucleic acid", "siNA", "short
interfering RNA", "siRNA", "short interfering nucleic acid
molecule", "short interfering oligonucleotide molecule", and
"chemically-modified short interfering nucleic acid molecule" as
used herein refer to any nucleic acid molecule capable of
inhibiting or down regulating gene expression or viral replication
by mediating RNA interference "RNAi" or gene silencing in a
sequence-specific manner (see PCT/US 2004/106390 (WO 05/19453),
U.S. Ser. No. 10/444,853, filed May 23, 2003 U.S. Ser. No.
10/923,536 filed Aug. 20, 2004, U.S. Ser. No. 11/234,730, filed
Sep. 23, 2005, U.S. Ser. No. 11/299,254, filed Dec. 8, 2005, or
PCT/US06/32168, filed Aug. 17, 2006, all incorporated by reference
in their entireties herein). These terms can refer to both
individual nucleic acid molecules, a plurality of such nucleic acid
molecules, or pools of such nucleic acid molecules. The siNA can be
a double-stranded nucleic acid molecule comprising
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be assembled from two
separate oligonucleotides, where one strand is the sense strand and
the other is the antisense strand, wherein the antisense and sense
strands are self-complementary (i.e., each strand comprises
nucleotide sequence that is complementary to nucleotide sequence in
the other strand; such as where the antisense strand and sense
strand form a duplex or double stranded structure, for example
wherein the double stranded region is about 15 to about 30, e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 base pairs; the antisense strand comprises nucleotide sequence
that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof (e.g., about 15 to about 25 or more
nucleotides of the siNA molecule are complementary to the target
nucleic acid or a portion thereof). Alternatively, the siNA is
assembled from a single oligonucleotide, where the
self-complementary sense and antisense regions of the siNA are
linked by means of a nucleic acid based or non-nucleic acid-based
linker(s). The siNA can be a polynucleotide with a duplex,
asymmetric duplex, hairpin or asymmetric hairpin secondary
structure, having self-complementary sense and antisense regions,
wherein the antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a separate target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The siNA can be a circular
single-stranded polynucleotide having two or more loop structures
and a stem comprising self-complementary sense and antisense
regions, wherein the antisense region comprises nucleotide sequence
that is complementary to nucleotide sequence in a target nucleic
acid molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof, and wherein the circular
polynucleotide can be processed either in vivo or in vitro to
generate an active siNA molecule capable of mediating RNAi. The
siNA can also comprise a single stranded polynucleotide having
nucleotide sequence complementary to nucleotide sequence in a
target nucleic acid molecule or a portion thereof (for example,
where such siNA molecule does not require the presence within the
siNA molecule of nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof), wherein the single
stranded polynucleotide can further comprise a terminal phosphate
group, such as a 5'-phosphate (see for example Martinez et al.,
2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell,
10, 537-568), or 5',3'-diphosphate. In certain embodiments, the
siNA molecule of the invention comprises separate sense and
antisense sequences or regions, wherein the sense and antisense
regions are covalently linked by nucleotide or non-nucleotide
linkers molecules as is known in the art, or are alternately
non-covalently linked by ionic interactions, hydrogen bonding, van
der waals interactions, hydrophobic interactions, and/or stacking
interactions. In certain embodiments, the siNA molecules of the
invention comprise nucleotide sequence that is complementary to
nucleotide sequence of a target gene. In another embodiment, the
siNA molecule of the invention interacts with nucleotide sequence
of a target gene in a manner that causes inhibition of expression
of the target gene. As used herein, siNA molecules need not be
limited to those molecules containing only RNA, but further
encompasses chemically-modified nucleotides and non-nucleotides. In
certain embodiments, the short interfering nucleic acid molecules
of the invention lack 2'-hydroxy (2'-OH) containing nucleotides.
Applicant describes in certain embodiments short interfering
nucleic acids that do not require the presence of nucleotides
having a 2'-hydroxy group for mediating RNAi and as such, short
interfering nucleic acid molecules of the invention optionally do
not include any ribonucleotides (e.g., nucleotides having a 2'-OH
group). Such siNA molecules that do not require the presence of
ribonucleotides within the siNA molecule to support RNAi can
however have an attached linker or linkers or other attached or
associated groups, moieties, or chains containing one or more
nucleotides with 2'-OH groups. Optionally, siNA molecules can
comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the
nucleotide positions. The modified short interfering nucleic acid
molecules of the invention can also be referred to as short
interfering modified oligonucleotides "siMON." As used herein, the
term siNA is meant to be equivalent to other terms used to describe
nucleic acid molecules that are capable of mediating sequence
specific RNAi, for example short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, short interfering
nucleic acid, short interfering modified oligonucleotide,
chemically-modified siRNA, post-transcriptional gene silencing RNA
(ptgsRNA), and others. Non limiting examples of siNA molecules of
the invention are shown in U.S. Ser. No. 11/234,730, filed Sep. 23,
2005, incorporated by reference in its entirety herein. Such siNA
molecules are distinct from other nucleic acid technologies known
in the art that mediate inhibition of gene expression, such as
ribozymes, antisense, triplex forming, aptamer, 2,5-A chimera, or
decoy oligonucleotides.
[0364] By "RNA interference" or "RNAi" is meant a biological
process of inhibiting or down regulating gene expression in a cell
as is generally known in the art and which is mediated by short
interfering nucleic acid molecules, see for example Zamore and
Haley, 2005, Science, 309, 1519-1524; Vaughn and Martienssen, 2005,
Science, 309, 1525-1526; Zamore et al., 2000, Cell, 101, 25-33;
Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature,
411, 494-498; and Kreutzer et al., International PCT Publication
No. WO 00/44895; Zernicka-Goetz et al., International PCT
Publication No. WO 01/36646; Fire, International PCT Publication
No. WO 99/32619; Plaetinck et al., International PCT Publication
No. WO 00/01846; Mello and Fire, International PCT Publication No.
WO 01/29058; Deschamps-Depaillette, International PCT Publication
No. WO 99/07409; and Li et al., International PCT Publication No.
WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,
2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297,
2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;
Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al.,
2002, RNA, 8, 842-850; Reinhart et al., 2002, gene & Dev., 16,
1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). In
addition, as used herein, the term RNAi is meant to be equivalent
to other terms used to describe sequence specific RNA interference,
such as post transcriptional gene silencing, translational
inhibition, transcriptional inhibition, or epigenetics. For
example, siNA molecules of the invention can be used to
epigenetically silence genes at both the post-transcriptional level
or the pre-transcriptional level. In a non-limiting example,
epigenetic modulation of gene expression by siNA molecules of the
invention can result from siNA mediated modification of chromatin
structure or methylation patterns to alter gene expression (see,
for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra
et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237). In another non-limiting example, modulation of gene
expression by siNA molecules of the invention can result from siNA
mediated cleavage of RNA (either coding or non-coding RNA) via
RISC, or alternately, translational inhibition as is known in the
art. In another embodiment, modulation of gene expression by siNA
molecules of the invention can result from transcriptional
inhibition (see for example Janowski et al., 2005, Nature Chemical
Biology, 1, 216-222).
[0365] By "asymmetric hairpin" as used herein is meant a linear
siNA molecule comprising an antisense region, a loop portion that
can comprise nucleotides or non-nucleotides, and a sense region
that comprises fewer nucleotides than the antisense region to the
extent that the sense region has enough complementary nucleotides
to base pair with the antisense region and form a duplex with loop.
For example, an asymmetric hairpin siNA molecule of the invention
can comprise an antisense region having length sufficient to
mediate RNAi in a cell or in vitro system (e.g. about 15 to about
30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 nucleotides) and a loop region comprising about 4 to
about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides,
and a sense region having about 3 to about 25 (e.g., about 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25) nucleotides that are complementary to the antisense
region. The asymmetric hairpin siNA molecule can also comprise a
5'-terminal phosphate group that can be chemically modified. The
loop portion of the asymmetric hairpin siNA molecule can comprise
nucleotides, non-nucleotides, linker molecules, or conjugate
molecules as described herein.
[0366] By "asymmetric duplex" as used herein is meant a siNA
molecule having two separate strands comprising a sense region and
an antisense region, wherein the sense region comprises fewer
nucleotides than the antisense region to the extent that the sense
region has enough complementary nucleotides to base pair with the
antisense region and form a duplex. For example, an asymmetric
duplex siNA molecule of the invention can comprise an antisense
region having length sufficient to mediate RNAi in a cell or in
vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and
a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25) nucleotides that are complementary to the antisense
region.
[0367] The term "polynucleotide" or "nucleic acid molecule" as used
herein, refers to a molecule having nucleotides. The nucleic acid
can be single, double, or multiple stranded and can comprise
modified or unmodified nucleotides or non-nucleotides or various
mixtures and combinations thereof.
[0368] By "RNAi inhibitor" is meant any molecule that can down
regulate, reduce or inhibit RNA interference function or activity
in a cell or organism. An RNAi inhibitor can down regulate, reduce
or inhibit RNAi (e.g., RNAi mediated cleavage of a target
polynucleotide, translational inhibition, or transcriptional
silencing) by interaction with or interfering the function of any
component of the RNAi pathway, including protein components such as
RISC, or nucleic acid components such as miRNAs or siRNAs. A RNAi
inhibitor can be a siNA molecule, an antisense molecule, an
aptamer, or a small molecule that interacts with or interferes with
the function of RISC, a miRNA, or a siRNA or any other component of
the RNAi pathway in a cell or organism. By inhibiting RNAi (e.g.,
RNAi mediated cleavage of a target polynucleotide, translational
inhibition, or transcriptional silencing), a RNAi inhibitor of the
invention can be used to modulate (e.g, up-regulate or down
regulate) the expression of a target gene. In one embodiment, a RNA
inhibitor of the invention is used to up-regulate gene expression
by interfering with (e.g., reducing or preventing) endogenous
down-regulation or inhibition of gene expression through
translational inhibition, transcriptional silencing, or RISC
mediated cleavage of a polynucleotide (e.g., mRNA). By interfering
with mechanisms of endogenous repression, silencing, or inhibition
of gene expression, RNAi inhibitors of the invention can therefore
be used to up-regulate gene expression for the treatment of
diseases, traits, or conditions resulting from a loss of function.
In one embodiment, the term "RNAi inhibitor" is used in place of
the term "siNA" in the various embodiments herein, for example,
with the effect of increasing gene expression for the treatment of
loss of function diseases, traits, and/or conditions.
[0369] The term "enzymatic nucleic acid molecule" as used herein
refers to a nucleic acid molecule which has complementarity in a
substrate binding region to a specified gene target, and also has
an enzymatic activity which is active to specifically cleave target
RNA. That is, the enzymatic nucleic acid molecule is able to
intermolecularly cleave RNA and thereby inactivate a target RNA
molecule. These complementary regions allow sufficient
hybridization of the enzymatic nucleic acid molecule to the target
RNA and thus permit cleavage. One hundred percent complementarity
is preferred, but complementarity as low as 50-75% can also be
useful in this invention (see for example Werner and Uhlenbeck,
1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,
Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids
can be modified at the base, sugar, and/or phosphate groups. The
term enzymatic nucleic acid is used interchangeably with phrases
such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA,
aptazyme or aptamer-binding ribozyme, regulatable ribozyme,
catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme,
endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or
DNA enzyme. All of these terminologies describe nucleic acid
molecules with enzymatic activity. The specific enzymatic nucleic
acid molecules described in the instant application are not
limiting in the invention and those skilled in the art will
recognize that all that is important in an enzymatic nucleic acid
molecule of this invention is that it has a specific substrate
binding site which is complementary to one or more of the target
nucleic acid regions, and that it have nucleotide sequences within
or surrounding that substrate binding site which impart a nucleic
acid cleaving and/or ligation activity to the molecule (Cech et
al., U.S. Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
Ribozymes and enzymatic nucleic molecules of the invention can be
chemically modified as is generally known in the art or as
described herein.
[0370] The term "antisense nucleic acid", as used herein, refers to
a non-enzymatic nucleic acid molecule that binds to target RNA by
means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid;
Egholm et al., 1993 Nature 365, 566) interactions and alters the
activity of the target RNA (for a review, see Stein and Cheng, 1993
Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902).
Typically, antisense molecules are complementary to a target
sequence along a single contiguous sequence of the antisense
molecule. However, in certain embodiments, an antisense molecule
can bind to substrate such that the substrate molecule forms a
loop, and/or an antisense molecule can bind such that the antisense
molecule forms a loop. Thus, the antisense molecule can be
complementary to two (or even more) non-contiguous substrate
sequences or two (or even more) non-contiguous sequence portions of
an antisense molecule can be complementary to a target sequence or
both. For a review of current antisense strategies, see Schmajuk et
al., 1999, J. Biol. Chem., 274, 21783-21789, Delihas et al., 1997,
Nature, 15, 751-753, Stein et al., 1997, Antisense N. A. Drug Dev.,
7, 151, Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998,
Biotech. Genet. Eng. Rev., 15, 121-157, Crooke, 1997, Ad.
Pharmacol., 40, 1-49. In addition, antisense DNA can be used to
target RNA by means of DNA-RNA interactions, thereby activating
RNase H, which digests the target RNA in the duplex. The antisense
oligonucleotides can comprise one or more RNAse H activating
region, which is capable of activating RNAse H cleavage of a target
RNA. Antisense DNA can be synthesized chemically or expressed via
the use of a single stranded DNA expression vector or equivalent
thereof. Antisense molecules of the invention can be chemically
modified as is generally known in the art or as described
herein.
[0371] The term "RNase H activating region" as used herein, refers
to a region (generally greater than or equal to 4-25 nucleotides in
length, preferably from 5-11 nucleotides in length) of a nucleic
acid molecule capable of binding to a target RNA to form a
non-covalent complex that is recognized by cellular RNase H enzyme
(see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et
al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to the
nucleic acid molecule-target RNA complex and cleaves the target RNA
sequence. The RNase H activating region comprises, for example,
phosphodiester, phosphorothioate (preferably at least four of the
nucleotides are phosphorothiote substitutions; more specifically,
4-11 of the nucleotides are phosphorothiote substitutions);
phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone
chemistry or a combination thereof. In addition to one or more
backbone chemistries described above, the RNase H activating region
can also comprise a variety of sugar chemistries. For example, the
RNase H activating region can comprise deoxyribose, arabino,
fluoroarabino or a combination thereof, nucleotide sugar chemistry.
Those skilled in the art will recognize that the foregoing are
non-limiting examples and that any combination of phosphate, sugar
and base chemistry of a nucleic acid that supports the activity of
RNase H enzyme is within the scope of the definition of the RNase H
activating region and the instant invention.
[0372] The term "2-5A antisense chimera" as used herein, refers to
an antisense oligonucleotide containing a 5'-phosphorylated
2'-5'-linked adenylate residue. These chimeras bind to target RNA
in a sequence-specific manner and activate a cellular
2-5A-dependent ribonuclease which, in turn, cleaves the target RNA
(Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300;
Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and
Torrence, 1998, Pharmacol. Ther., 78, 55-113). 2-5A antisense
chimera molecules of the invention can be chemically modified as is
generally known in the art or as described herein.
[0373] The term "triplex forming oligonucleotides" as used herein,
refers to an oligonucleotide that can bind to a double-stranded DNA
in a sequence-specific manner to form a triple-strand helix.
Formation of such triple helix structure has been shown to inhibit
transcription of the targeted gene (Duval-Valentin et al., 1992
Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7,
17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489,
181-206). Triplex forming oligonucleotide molecules of the
invention can be chemically modified as is generally known in the
art or as described herein.
[0374] The term "decoy RNA" as used herein, refers to a RNA
molecule or aptamer that is designed to preferentially bind to a
predetermined ligand. Such binding can result in the inhibition or
activation of a target molecule. The decoy RNA or aptamer can
compete with a naturally occurring binding target for the binding
of a specific ligand. For example, it has been shown that
over-expression of HIV trans-activation response (TAR) RNA can act
as a "decoy" and efficiently binds HIV tat protein, thereby
preventing it from binding to TAR sequences encoded in the HIV RNA
(Sullenger et al., 1990, Cell, 63, 601-608). This is but a specific
example and those in the art will recognize that other embodiments
can be readily generated using techniques generally known in the
art, see for example Gold et al., 1995, Annu. Rev. Biochem., 64,
763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr.
Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27;
Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999,
Clinical Chemistry, 45, 1628. Similarly, a decoy RNA can be
designed to bind to a receptor and block the binding of an effector
molecule or a decoy RNA can be designed to bind to receptor of
interest and prevent interaction with the receptor. Decoy molecules
of the invention can be chemically modified as is generally known
in the art or as described herein.
[0375] The term "single stranded RNA" (ssRNA) as used herein refers
to a naturally occurring or synthetic ribonucleic acid molecule
comprising a linear single strand, for example a ssRNA can be a
messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA)
etc. of a gene.
[0376] The term "single stranded DNA" (ssDNA) as used herein refers
to a naturally occurring or synthetic deoxyribonucleic acid
molecule comprising a linear single strand, for example, a ssDNA
can be a sense or antisense gene sequence or EST (Expressed
Sequence Tag).
[0377] The term "double stranded RNA" or "dsRNA" as used herein
refers to a double stranded RNA molecule capable of RNA
interference, including short interfering RNA (siNA).
[0378] The term "allozyme" as used herein refers to an allosteric
enzymatic nucleic acid molecule, see for example see for example
George et al., U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al.,
U.S. Pat. No. 5,589,332, Nathan et al., U.S. Pat. No 5,871,914,
Nathan and Ellington, International PCT publication No. WO
00/24931, Breaker et al., International PCT Publication Nos. WO
00/26226 and 98/27104, and Sullenger et al., International PCT
publication No. WO 99/29842.
[0379] By "aptamer" or "nucleic acid aptamer" as used herein is
meant a polynucleotide that binds specifically to a target molecule
wherein the nucleic acid molecule has sequence that is distinct
from sequence recognized by the target molecule in its natural
setting. Alternately, an aptamer can be a nucleic acid molecule
that binds to a target molecule where the target molecule does not
naturally bind to a nucleic acid. The target molecule can be any
molecule of interest. For example, the aptamer can be used to bind
to a ligand-binding domain of a protein, thereby preventing
interaction of the naturally occurring ligand with the protein.
This is a non-limiting example and those in the art will recognize
that other embodiments can be readily generated using techniques
generally known in the art, see for example Gold et al., 1995,
Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol.,
74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J.
Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287, 820;
and Jayasena, 1999, Clinical Chemistry, 45, 1628. Aptamer molecules
of the invention can be chemically modified as is generally known
in the art or as described herein.
[0380] By "modulate" is meant that the expression of the gene, or
level of RNA molecule or equivalent RNA molecules encoding one or
more proteins or protein subunits, or activity of one or more
proteins or protein subunits is up regulated or down regulated,
such that expression, level, or activity is greater than or less
than that observed in the absence of the modulator. For example,
the term "modulate" can mean "inhibit," but the use of the word
"modulate" is not limited to this definition.
[0381] By "inhibit", "down-regulate", or "reduce", it is meant that
the expression of the gene, or level of RNA molecules or equivalent
RNA molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is reduced
below that observed in the absence of the nucleic acid molecules
(e.g., siNA) of the invention. In one embodiment, inhibition,
down-regulation or reduction with a siNA molecule is below that
level observed in the presence of an inactive or attenuated
molecule. In another embodiment, inhibition, down-regulation, or
reduction with siNA molecules is below that level observed in the
presence of, for example, a siNA molecule with scrambled sequence
or with mismatches. In another embodiment, inhibition,
down-regulation, or reduction of gene expression with a nucleic
acid molecule of the instant invention is greater in the presence
of the nucleic acid molecule than in its absence. In one
embodiment, inhibition, down regulation, or reduction of gene
expression is associated with post transcriptional silencing, such
as RNAi mediated cleavage of a target nucleic acid molecule (e.g.
RNA) or inhibition of translation. In one embodiment, inhibition,
down regulation, or reduction of gene expression is associated with
pretranscriptional silencing.
[0382] By "up-regulate", or "promote", it is meant that the
expression of the gene, or level of RNA molecules or equivalent RNA
molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is increased
above that observed in the absence of the nucleic acid molecules
(e.g., siNA) of the invention. In one embodiment, up-regulation or
promotion of gene expression with an siNA molecule is above that
level observed in the presence of an inactive or attenuated
molecule. In another embodiment, up-regulation or promotion of gene
expression with siNA molecules is above that level observed in the
presence of, for example, an siNA molecule with scrambled sequence
or with mismatches. In another embodiment, up-regulation or
promotion of gene expression with a nucleic acid molecule of the
instant invention is greater in the presence of the nucleic acid
molecule than in its absence. In one embodiment, up-regulation or
promotion of gene expression is associated with inhibition of RNA
mediated gene silencing, such as RNAi mediated cleavage or
silencing of a coding or non-coding RNA target that down regulates,
inhibits, or silences the expression of the gene of interest to be
up-regulated. The down regulation of gene expression can, for
example, be induced by a coding RNA or its encoded protein, such as
through negative feedback or antagonistic effects. The down
regulation of gene expression can, for example, be induced by a
non-coding RNA having regulatory control over a gene of interest,
for example by silencing expression of the gene via translational
inhibition, chromatin structure, methylation, RISC mediated RNA
cleavage, or translational inhibition. As such, inhibition or down
regulation of targets that down regulate, suppress, or silence a
gene of interest can be used to up-regulate or promote expression
of the gene of interest toward therapeutic use.
[0383] In one embodiment, a RNAi inhibitor of the invention is used
to up regulate gene expression by inhibiting RNAi or gene
silencing. For example, a RNAi inhibitor of the invention can be
used to treat loss of function diseases and conditions by
up-regulating gene expression, such as in instances of
haploinsufficiency where one allele of a particular gene harbors a
mutation (e.g., a frameshift, missense, or nonsense mutation)
resulting in a loss of function of the protein encoded by the
mutant allele. In such instances, the RNAi inhibitor can be used to
up regulate expression of the protein encoded by the wild type or
functional allele, thus correcting the haploinsufficiency by
compensating for the mutant or null allele. In another embodiment,
a siNA molecule of the invention is used to down regulate
expression of a toxic gain of function allele while a RNAi
inhibitor of the invention is used concomitantly to up regulate
expression of the wild type or functional allele, such as in the
treatment of diseases, traits, or conditions herein or otherwise
known in the art (see for example Rhodes et al., 2004, PNAS USA,
101:11147-11152 and Meisler et al. 2005, The Journal of Clinical
Investigation, 115:2010-2017).
[0384] By "gene", or "target gene", is meant a nucleic acid that
encodes RNA, for example, nucleic acid sequences including, but not
limited to, structural genes encoding a polypeptide. A gene or
target gene can also encode a functional RNA (fRNA) or non-coding
RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small
nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)
and precursor RNAs thereof. Such non-coding RNAs can serve as
target nucleic acid molecules for siNA mediated RNA interference in
modulating the activity of fRNA or ncRNA involved in functional or
regulatory cellular processes. Abberant fRNA or ncRNA activity
leading to disease can therefore be modulated by siNA molecules of
the invention. siNA molecules targeting fRNA and ncRNA can also be
used to manipulate or alter the genotype or phenotype of a subject,
organism or cell, by intervening in cellular processes such as
genetic imprinting, transcription, translation, or nucleic acid
processing (e.g., transamination, methylation etc.). The target
gene can be a gene derived from a cell, an endogenous gene, a
transgene, or exogenous genes such as genes of a pathogen, for
example a virus, which is present in the cell after infection
thereof. The cell containing the target gene can be derived from or
contained in any organism, for example a plant, animal, protozoan,
virus, bacterium, or fungus. Non-limiting examples of plants
include monocots, dicots, or gymnosperms. Non-limiting examples of
animals include vertebrates or invertebrates. Non-limiting examples
of fungi include molds or yeasts. For a review, see for example
Snyder and Gerstein, 2003, Science, 300, 258-260.
[0385] By "target" as used herein is meant, any target protein,
peptide, or polypeptide encoded by a target gene. The term "target"
also refers to nucleic acid sequences encoding any target protein,
peptide, or polypeptide having target activity, such as encoded by
target RNA. The term "target" is also meant to include other target
encoding sequence, such as other target isoforms, mutant target
genes, splice variants of target genes, and target gene
polymorphisms. By "target nucleic acid" is meant any nucleic acid
sequence whose expression or activity is to be modulated. The
target nucleic acid can be DNA or RNA.
[0386] By "non-canonical base pair" is meant any non-Watson Crick
base pair, such as mismatches and/or wobble base pairs, including
flipped mismatches, single hydrogen bond mismatches, trans-type
mismatches, triple base interactions, and quadruple base
interactions. Non-limiting examples of such non-canonical base
pairs include, but are not limited to, AC reverse Hoogsteen, AC
wobble, AU reverse Hoogsteen, GU wobble, AA N7 amino, CC
2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-amino,
UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AU reverse
Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA
N1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl,
GA+ carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino
symmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU
2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA
amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC
N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU
N7-imino, CC carbonyl-amino, GA amino-N1, GA amino-N7, GA
carbonyl-amino, GA N3-amino, GC amino-N3, GC carbonyl-amino, GC
N3-amino, GC N7-amino, GG amino-N7, GG carbonyl-imino, GG N7-amino,
GU amino-2-carbonyl, GU carbonyl-imino, GU imino-2-carbonyl, GU
N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC
imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GA carbonyl-C2-H,
UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A) N3(C)-carbonyl, GC
imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and
GU imino amino-2-carbonyl base pairs.
[0387] By "target" as used herein is meant, any target protein,
peptide, or polypeptide, such as encoded by Genbank Accession Nos.
shown in U.S. Ser. No. 10/923,536 and U.S. Ser. No. 10/923,536,
both incorporated by reference herein. The term "target" also
refers to nucleic acid sequences or target polynucleotide sequence
encoding any target protein, peptide, or polypeptide, such as
proteins, peptides, or polypeptides encoded by sequences having
Genbank Accession Nos. shown in U.S. Ser. No. 10/923,536 and U.S.
Ser. No. 10/923,536. The target of interest can include target
polynucleotide sequences, such as target DNA or target RNA. The
term "target" is also meant to include other sequences, such as
differing isoforms, mutant target genes, splice variants of target
polynucleotides, target polymorphisms, and non-coding (e.g., ncRNA,
miRNA, sRNA) or other regulatory polynucleotide sequences as
described herein. Therefore, in various embodiments of the
invention, a double stranded nucleic acid molecule of the invention
(e.g., siNA) having complementarity to a target RNA can be used to
inhibit or down regulate miRNA or other ncRNA activity. In one
embodiment, inhibition of miRNA or ncRNA activity can be used to
down regulate or inhibit gene expression (e.g., gene targets
described herein or otherwise known in the art) or viral
replication (e.g., viral targets described herein or otherwise
known in the art) that is dependent on miRNA or ncRNA activity. In
another embodiment, inhibition of miRNA or ncRNA activity by double
stranded nucleic acid molecules of the invention (e.g. siNA) having
complementarity to the miRNA or ncRNA can be used to up regulate or
promote target gene expression (e.g., gene targets described herein
or otherwise known in the art) where the expression of such genes
is down regulated, suppressed, or silenced by the miRNA or ncRNA.
Such up-regulation of gene expression can be used to treat diseases
and conditions associated with a loss of function or
haploinsufficiency as are generally known in the art (e.g.,
muscular dystrophies, cystic fibrosis, or neurologic diseases and
conditions described herein such as epilepsy, including severe
myoclonic epilepsy of infancy or Dravet syndrome).
[0388] By "homologous sequence" is meant, a nucleotide sequence
that is shared by one or more polynucleotide sequences, such as
genes, gene transcripts and/or non-coding polynucleotides. For
example, a homologous sequence can be a nucleotide sequence that is
shared by two or more genes encoding related but different
proteins, such as different members of a gene family, different
protein epitopes, different protein isoforms or completely
divergent genes, such as a cytokine and its corresponding
receptors. A homologous sequence can be a nucleotide sequence that
is shared by two or more non-coding polynucleotides, such as
noncoding DNA or RNA, regulatory sequences, introns, and sites of
transcriptional control or regulation. Homologous sequences can
also include conserved sequence regions shared by more than one
polynucleotide sequence. Homology does not need to be perfect
homology (e.g., 100%), as partially homologous sequences are also
contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%,
95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,
82%, 81%, 80% etc.).
[0389] By "conserved sequence region" is meant, a nucleotide
sequence of one or more regions in a polynucleotide does not vary
significantly between generations or from one biological system,
subject, or organism to another biological system, subject, or
organism. The polynucleotide can include both coding and non-coding
DNA and RNA.
[0390] By "sense region" is meant a nucleotide sequence of a siNA
molecule having complementarity to an antisense region of the siNA
molecule. In addition, the sense region of a siNA molecule can
comprise a nucleic acid sequence having homology with a target
nucleic acid sequence. In one embodiment, the sense region of the
siNA molecule is referred to as the sense strand or passenger
strand.
[0391] By "antisense region" is meant a nucleotide sequence of a
siNA molecule having complementarity to a target nucleic acid
sequence. In addition, the antisense region of a siNA molecule can
optionally comprise a nucleic acid sequence having complementarity
to a sense region of the siNA molecule. In one embodiment, the
antisense region of the siNA molecule is referred to as the
antisense strand or guide strand.
[0392] By "target nucleic acid" or "target polynucleotide" is meant
any nucleic acid sequence whose expression or activity is to be
modulated. The target nucleic acid can be DNA or RNA. In one
embodiment, a target nucleic acid of the invention is target RNA or
DNA.
[0393] By "complementarity" is meant that a nucleic acid can form
hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types as
described herein. In one embodiment, a double stranded nucleic acid
molecule of the invention, such as an siNA molecule, wherein each
strand is between 15 and 30 nucleotides in length, comprises
between about 10% and about 100% (e.g., about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100%) complementarity between the two
strands of the double stranded nucleic acid molecule. In another
embodiment, a double stranded nucleic acid molecule of the
invention, such as an siNA molecule, where one strand is the sense
strand and the other stand is the antisense strand, wherein each
strand is between 15 and 30 nucleotides in length, comprises
between at least about 10% and about 100% (e.g., at least about
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%)
complementarity between the nucleotide sequence in the antisense
strand of the double stranded nucleic acid molecule and the
nucleotide sequence of its corresponding target nucleic acid
molecule, such as a target RNA or target mRNA or viral RNA. In one
embodiment, a double stranded nucleic acid molecule of the
invention, such as an siNA molecule, where one strand comprises
nucleotide sequence that is referred to as the sense region and the
other strand comprises a nucleotide sequence that is referred to as
the antisense region, wherein each strand is between 15 and 30
nucleotides in length, comprises between about 10% and about 100%
(e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%)
complementarity between the sense region and the antisense region
of the double stranded nucleic acid molecule. In reference to the
nucleic molecules of the present invention, the binding free energy
for a nucleic acid molecule with its complementary sequence is
sufficient to allow the relevant function of the nucleic acid to
proceed, e.g., RNAi activity. Determination of binding free
energies for nucleic acid molecules is well known in the art (see,
e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133;
Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner
et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percent
complementarity indicates the percentage of contiguous residues in
a nucleic acid molecule that can form hydrogen bonds (e.g.,
Watson-Crick base pairing) with a second nucleic acid sequence
(e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10
nucleotides in the first oligonucleotide being based paired to a
second nucleic acid sequence having 10 nucleotides represents 50%,
60%, 70%, 80%, 90%, and 100% complementary respectively). In one
embodiment, a siNA molecule of the invention has perfect
complementarity between the sense strand or sense region and the
antisense strand or antisense region of the siNA molecule. In one
embodiment, a siNA molecule of the invention is perfectly
complementary to a corresponding target nucleic acid molecule.
"Perfectly complementary" means that all the contiguous residues of
a nucleic acid sequence will hydrogen bond with the same number of
contiguous residues in a second nucleic acid sequence. In one
embodiment, a siNA molecule of the invention comprises about 15 to
about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 or more) nucleotides that are
complementary to one or more target nucleic acid molecules or a
portion thereof. In one embodiment, a siNA molecule of the
invention has partial complementarity (i.e., less than 100%
complementarity) between the sense strand or sense region and the
antisense strand or antisense region of the siNA molecule or
between the antisense strand or antisense region of the siNA
molecule and a corresponding target nucleic acid molecule. For
example, partial complementarity can include various mismatches or
non-based paired nucleotides (e.g., 1, 2, 3, 4, 5 or more
mismatches or non-based paired nucleotides) within the siNA
structure which can result in bulges, loops, or overhangs that
result between the between the sense strand or sense region and the
antisense strand or antisense region of the siNA molecule or
between the antisense strand or antisense region of the siNA
molecule and a corresponding target nucleic acid molecule.
[0394] In one embodiment, a double stranded nucleic acid molecule
of the invention, such as siNA molecule, has perfect
complementarity between the sense strand or sense region and the
antisense strand or antisense region of the nucleic acid molecule.
In one embodiment, double stranded nucleic acid molecule of the
invention, such as siNA molecule, is perfectly complementary to a
corresponding target nucleic acid molecule.
[0395] In one embodiment, double stranded nucleic acid molecule of
the invention, such as siNA molecule, has partial complementarity
(i.e., less than 100% complementarity) between the sense strand or
sense region and the antisense strand or antisense region of the
double stranded nucleic acid molecule or between the antisense
strand or antisense region of the nucleic acid molecule and a
corresponding target nucleic acid molecule. For example, partial
complementarity can include various mismatches or non-base paired
nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based
paired nucleotides, such as nucleotide bulges) within the double
stranded nucleic acid molecule, structure which can result in
bulges, loops, or overhangs that result between the sense strand or
sense region and the antisense strand or antisense region of the
double stranded nucleic acid molecule or between the antisense
strand or antisense region of the double stranded nucleic acid
molecule and a corresponding target nucleic acid molecule.
[0396] In one embodiment, double stranded nucleic acid molecule of
the invention is a microRNA (miRNA). By "microRNA" or "miRNA" is
meant, a small double stranded RNA that regulates the expression of
target messenger RNAs either by mRNA cleavage, translational
repression/inhibition or heterochromatic silencing (see for example
Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116,
281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004,
Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342,
25-28). In one embodiment, the microRNA of the invention, has
partial complementarity (i.e., less than 100% complementarity)
between the sense strand or sense region and the antisense strand
or antisense region of the miRNA molecule or between the antisense
strand or antisense region of the miRNA and a corresponding target
nucleic acid molecule. For example, partial complementarity can
include various mismatches or non-base paired nucleotides (e.g., 1,
2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such
as nucleotide bulges) within the double stranded nucleic acid
molecule, structure which can result in bulges, loops, or overhangs
that result between the sense strand or sense region and the
antisense strand or antisense region of the miRNA or between the
antisense strand or antisense region of the miRNA and a
corresponding target nucleic acid molecule.
[0397] In one embodiment, compositions of the invention such as
formulated molecular compositions and formulated siNA compositions
of the invention that down regulate or reduce target gene
expression are used for preventing or treating diseases, disorders,
conditions, or traits in a subject or organism as described herein
or otherwise known in the art.
[0398] By "proliferative disease" or "cancer" as used herein is
meant, any disease, condition, trait, genotype or phenotype
characterized by unregulated cell growth or replication as is known
in the art; including leukemias, for example, acute myelogenous
leukemia (AML), chronic myelogenous leukemia (CML), acute
lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS
related cancers such as Kaposi's sarcoma; breast cancers; bone
cancers such as Osteosarcoma, Chondrosarcomas, Ewing's sarcoma,
Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas;
Brain cancers such as Meningiomas, Glioblastomas, Lower-Grade
Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas,
and Metastatic brain cancers; cancers of the head and neck
including various lymphomas such as mantle cell lymphoma,
non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngeal
carcinoma, gallbladder and bile duct cancers, cancers of the retina
such as retinoblastoma, cancers of the esophagus, gastric cancers,
multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer,
testicular cancer, endometrial cancer, melanoma, colorectal cancer,
lung cancer, bladder cancer, prostate cancer, lung cancer
(including non-small cell lung carcinoma), pancreatic cancer,
sarcomas, Wilms' tumor, cervical cancer, head and neck cancer, skin
cancers, nasopharyngeal carcinoma, liposarcoma, epithelial
carcinoma, renal cell carcinoma, gallbladder adeno carcinoma,
parotid adenocarcinoma, endometrial sarcoma, multidrug resistant
cancers; and proliferative diseases and conditions, such as
neovascularization associated with tumor angiogenesis, macular
degeneration (e.g., wet/dry AMD), corneal neovascularization,
diabetic retinopathy, neovascular glaucoma, myopic degeneration and
other proliferative diseases and conditions such as restenosis and
polycystic kidney disease, and any other cancer or proliferative
disease, condition, trait, genotype or phenotype that can respond
to the modulation of disease related gene expression in a cell or
tissue, alone or in combination with other therapies.
[0399] By "inflammatory disease" or "inflammatory condition" as
used herein is meant any disease, condition, trait, genotype or
phenotype characterized by an inflammatory or allergic process as
is known in the art, such as inflammation, acute inflammation,
chronic inflammation, respiratory disease, atherosclerosis,
psoriasis, dermatitis, restenosis, asthma, allergic rhinitis,
atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory
bowl disease, inflammotory pelvic disease, pain, ocular
inflammatory disease, celiac disease, Leigh Syndrome, Glycerol
Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive
spastic ataxia, laryngeal inflammatory disease; Tuberculosis,
Chronic cholecystitis, Bronchiectasis, Silicosis and other
pneumoconioses, and any other inflammatory disease, condition,
trait, genotype or phenotype that can respond to the modulation of
disease related gene expression in a cell or tissue, alone or in
combination with other therapies.
[0400] By "autoimmune disease" or "autoimmune condition" as used
herein is meant, any disease, condition, trait, genotype or
phenotype characterized by autoimmunity as is known in the art,
such as multiple sclerosis, diabetes mellitus, lupus, celiac
disease, Crohn's disease, ulcerative colitis, Guillain-Barre
syndrome, scleroderms, Goodpasture's syndrome, Wegener's
granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis,
Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune
hepatitis, Addison's disease, Hashimoto's thyroiditis,
Fibromyalgia, Menier's syndrome; transplantation rejection (e.g.,
prevention of allograft rejection) pernicious anemia, rheumatoid
arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, Reiter's syndrome, Grave's disease, and any other
autoimmune disease, condition, trait, genotype or phenotype that
can respond to the modulation of disease related gene expression in
a cell or tissue, alone or in combination with other therapies.
[0401] By "infectious disease" is meant any disease, condition,
trait, genotype or phenotype associated with an infectious agent,
such as a virus, bacteria, fungus, prion, or parasite. Non-limiting
examples of various viral genes that can be targeted using siNA
molecules of the invention include Hepatitis C Virus (HCV, for
example Genbank Accession Nos: D11168, D50483.1, L38318 and
S82227), Hepatitis B Virus (HBV, for example GenBank Accession No.
AF100308.1), Human Immunodeficiency Virus type 1 (HIV-1, for
example GenBank Accession No. U51188), Human Immunodeficiency Virus
type 2 (HIV-2, for example GenBank Accession No. X60667), West Nile
Virus (WNV for example GenBank accession No. NC.sub.--001563),
cytomegalovirus (CMV for example GenBank Accession No.
NC.sub.--001347), respiratory syncytial virus (RSV for example
GenBank Accession No. NC.sub.--001781), influenza virus (for
example GenBank Accession No. AF037412, rhinovirus (for example,
GenBank accession numbers: D00239, X02316, X01087, L24917, M16248,
K02121, X01087), papillomavirus (for example GenBank Accession No.
NC.sub.--001353), Herpes Simplex Virus (HSV for example GenBank
Accession No. NC.sub.--001345), and other viruses such as HTLV (for
example GenBank Accession No. AJ430458). Due to the high sequence
variability of many viral genomes, selection of siNA molecules for
broad therapeutic applications would likely involve the conserved
regions of the viral genome. Nonlimiting examples of conserved
regions of the viral genomes include but are not limited to 5'-Non
Coding Regions (NCR), 3'-Non Coding Regions (NCR) and/or internal
ribosome entry sites (IRES). siNA molecules designed against
conserved regions of various viral genomes will enable efficient
inhibition of viral replication in diverse patient populations and
may ensure the effectiveness of the siNA molecules against viral
quasi species which evolve due to mutations in the non-conserved
regions of the viral genome. Non-limiting examples of bacterial
infections include Actinomycosis, Anthrax, Aspergillosis,
Bacteremia, Bacterial Infections and Mycoses, Bartonella
Infections, Botulism, Brucellosis, Burkholderia Infections,
Campylobacter Infections, Candidiasis, Cat-Scratch Disease,
Chlamydia Infections, Cholera, Clostridium Infections,
Coccidioidomycosis, Cross Infection, Cryptococcosis,
Dermatomycoses, Dermatomycoses, Diphtheria, Ehrlichiosis,
Escherichia coli Infections, Fasciitis, Necrotizing, Fusobacterium
Infections, Gas Gangrene, Gram-Negative Bacterial Infections,
Gram-Positive Bacterial Infections, Histoplasmosis, Impetigo,
Klebsiella Infections, Legionellosis, Leprosy, Leptospirosis,
Listeria Infections, Lyme Disease, Maduromycosis, Melioidosis,
Mycobacterium Infections, Mycoplasma Infections, Mycoses, Nocardia
Infections, Onychomycosis, Ornithosis, Plague, Pneumococcal
Infections, Pseudomonas Infections, Q Fever, Rat-Bite Fever,
Relapsing Fever, Rheumatic Fever, Rickettsia Infections, Rocky
Mountain Spotted Fever, Salmonella Infections, Scarlet Fever, Scrub
Typhus, Sepsis, Sexually Transmitted Diseases--Bacterial, Bacterial
Skin Diseases, Staphylococcal Infections, Streptococcal Infections,
Tetanus, Tick-Borne Diseases, Tuberculosis, Tularemia, Typhoid
Fever, Typhus, Epidemic Louse-Borne, Vibrio Infections, Yaws,
Yersinia Infections, Zoonoses, and Zygomycosis. Non-limiting
examples of fungal infections include Aspergillosis, Blastomycosis,
Coccidioidomycosis, Cryptococcosis, Fungal Infections of
Fingernails and Toenails, Fungal Sinusitis, Histoplasmosis,
Histoplasmosis, Mucormycosis, Nail Fungal Infection,
Paracoccidioidomycosis, Sporotrichosis, Valley Fever
(Coccidioidomycosis), and Mold Allergy.
[0402] By "neurologic disease" or "neurological disease" is meant
any disease, disorder, or condition affecting the central or
peripheral nervous system, inlcuding ADHD, AIDS--Neurological
Complications, Absence of the Septum Pellucidum, Acquired
Epileptiform Aphasia, Acute Disseminated Encephalomyelitis,
Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia,
Aicardi Syndrome, Alexander Disease, Alpers' Disease, Alternating
Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis,
Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia,
Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari
Malformation, Arteriovenous Malformation, Aspartame, Asperger
Syndrome, Ataxia Telangiectasia, Ataxia, Attention
Deficit-Hyperactivity Disorder, Autism, Autonomic Dysfunction, Back
Pain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's
Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy,
Benign Intracranial Hypertension, Bernhardt-Roth Syndrome,
Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome,
Brachial Plexus Birth Injuries, Brachial Plexus Injuries,
Bradbury-Eggleston Syndrome, Brain Aneurysm, Brain Injury, Brain
and Spinal Tumors, Brown-Sequard Syndrome, Bulbospinal Muscular
Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia,
Cavernomas, Cavernous Angioma, Cavernous Malformation, Central
Cervical Cord Syndrome, Central Cord Syndrome, Central Pain
Syndrome, Cephalic Disorders, Cerebellar Degeneration, Cerebellar
Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral
Atrophy, Cerebral Beriberi, Cerebral Gigantism, Cerebral Hypoxia,
Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome,
Charcot-Marie-Tooth Disorder, Chiari Malformation, Chorea,
Choreoacanthocytosis, Chronic Inflammatory Demyelinating
Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic
Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma,
including Persistent Vegetative State, Complex Regional Pain
Syndrome, Congenital Facial Diplegia, Congenital Myasthenia,
Congenital Myopathy, Congenital Vascular Cavernous Malformations,
Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis,
Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's
Syndrome, Cytomegalic Inclusion Body Disease (CIBD),
Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome,
Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome,
Dejerine-Klumpke Palsy, Dementia--Multi-Infarct,
Dementia--Subcortical, Dementia With Lewy Bodies, Dermatomyositis,
Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy,
Diffuse Sclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia,
Dyslexia, Dysphagia, Dyspraxia, Dystonias, Early Infantile
Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis
Lethargica, Encephalitis and Meningitis, Encephaloceles,
Encephalopathy, Encephalotrigeminal Angiomatosis, Epilepsy, Erb's
Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Fabry's Disease,
Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial
Hemangioma, Familial Idiopathic Basal Ganglia Calcification,
Familial Spastic Paralysis, Febrile Seizures (e.g., GEFS and GEFS
plus), Fisher Syndrome, Floppy Infant Syndrome, Friedreich's
Ataxia, Gaucher's Disease, Gerstmann's Syndrome,
Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant
Cell Inclusion Disease, Globoid Cell Leukodystrophy,
Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1
Associated Myelopathy, Hallervorden-Spatz Disease, Head Injury,
Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia
Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia,
Heredopathia Atactica Polyneuritiformis, Herpes Zoster Oticus,
Herpes Zoster, Hirayama Syndrome, Holoprosencephaly, Huntington's
Disease, Hydranencephaly, Hydrocephalus--Normal Pressure,
Hydrocephalus, Hydromyelia, Hypercortisolism, Hypersomnia,
Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis,
Inclusion Body Myositis, Incontinentia Pigmenti, Infantile
Hypotonia, Infantile Phytanic Acid Storage Disease, Infantile
Refsum Disease, Infantile Spasms, Inflammatory Myopathy, Intestinal
Lipodystrophy, Intracranial Cysts, Intracranial Hypertension,
Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome,
Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin syndrome,
Klippel Feil Syndrome, Klippel-Trenaunay Syndrome (KTS),
Kluver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, Krabbe Disease,
Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic
Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve
Entrapment, Lateral Medullary Syndrome, Learning Disabilities,
Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome,
Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia,
Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease,
Lupus--Neurological Sequelae, Lyme Disease--Neurological
Complications, Machado-Joseph Disease, Macrencephaly,
Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Menkes
Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy,
Microcephaly, Migraine, Miller Fisher Syndrome, Mini-Strokes,
Mitochondrial Myopathies, Mobius Syndrome, Monomelic Amyotrophy,
Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses,
Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor
Neuropathy, Multiple Sclerosis, Multiple System Atrophy with
Orthostatic Hypotension, Multiple System Atrophy, Muscular
Dystrophy, Myasthenia--Congenital, Myasthenia Gravis,
Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of
Infants, Myoclonus, Myopathy--Congenital, Myopathy--Thyrotoxic,
Myopathy, Myotonia Congenita, Myotonia, Narcolepsy,
Neuroacanthocytosis, Neurodegeneration with Brain Iron
Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,
Neurological Complications of AIDS, Neurological Manifestations of
Pompe Disease, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid
Lipofuscinosis, Neuronal Migration Disorders,
Neuropathy--Hereditary, Neurosarcoidosis, Neurotoxicity, Nevus
Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome,
Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara
Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus,
Orthostatic Hypotension, Overuse Syndrome, Pain--Chronic,
Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease,
Parmyotonia Congenita, Paroxysmal Choreoathetosis, Paroxysmal
Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena
Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses,
Peripheral Neuropathy, Periventricular Leukomalacia, Persistent
Vegetative State, Pervasive Developmental Disorders, Phytanic Acid
Storage Disease, Pick's Disease, Piriformis Syndrome, Pituitary
Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio
Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis,
Postural Hypotension, Postural Orthostatic Tachycardia Syndrome,
Postural Tachycardia Syndrome, Primary Lateral Sclerosis, Prion
Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor
Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive
Sclerosing Poliodystrophy, Progressive Supranuclear Palsy,
Pseudotumor Cerebri, Pyridoxine Dependent and Pyridoxine Responsive
Seizure Disorders, Ramsay Hunt Syndrome Type I, Ramsay Hunt
Syndrome Type II, Rasmussen's Encephalitis and other autoimmune
epilepsies, Reflex Sympathetic Dystrophy Syndrome, Refsum
Disease--Infantile, Refsum Disease, Repetitive Motion Disorders,
Repetitive Stress Injuries, Restless Legs Syndrome,
Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome,
Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint
Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's
Disease, Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia,
Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome,
Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea,
Sleeping Sickness, Soto's Syndrome, Spasticity, Spina Bifida,
Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors,
Spinal Muscular Atrophy, Spinocerebellar Atrophy,
Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome,
Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute
Sclerosing Panencephalitis, Subcortical Arteriosclerotic
Encephalopathy, Swallowing Disorders, Sydenham Chorea, Syncope,
Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia,
Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia,
Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered
Spinal Cord Syndrome, Thomsen Disease, Thoracic Outlet Syndrome,
Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette
Syndrome, Transient Ischemic Attack, Transmissible Spongiform
Encephalopathies, Transverse Myelitis, Traumatic Brain Injury,
Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis,
Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis including
Temporal Arteritis, Von Economo's Disease, Von Hippel-Lindau
disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome,
Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West
Syndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease,
X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger
Syndrome.
[0403] By "respiratory disease" is meant, any disease or condition
affecting the respiratory tract, such as asthma, chronic
obstructive pulmonary disease or "COPD", allergic rhinitis,
sinusitis, pulmonary vasoconstriction, inflammation, allergies,
impeded respiration, respiratory distress syndrome, cystic
fibrosis, pulmonary hypertension, pulmonary vasoconstriction,
emphysema, and any other respiratory disease, condition, trait,
genotype or phenotype that can respond to the modulation of disease
related gene expression in a cell or tissue, alone or in
combination with other therapies.
[0404] By "cardiovascular disease" is meant and disease or
condition affecting the heart and vasculature, inlcuding but not
limited to, coronary heart disease (CHD), cerebrovascular disease
(CVD), aortic stenosis, peripheral vascular disease,
atherosclerosis, arteriosclerosis, myocardial infarction (heart
attack), cerebrovascular diseases (stroke), transient ischaemic
attacks (TIA), angina (stable and unstable), atrial fibrillation,
arrhythmia, vascular disease, congestive heart failure,
hypercholoesterolemia, type I hyperlipoproteinemia, type II
hyperlipoproteinemia, type III hyperlipoproteinemia, type IV
hyperlipoproteinemia, type V hyperlipoproteinemia, secondary
hypertrigliceridemia, and familial lecithin cholesterol
acyltransferase deficiency.
[0405] By "ocular disease" as used herein is meant, any disease,
condition, trait, genotype or phenotype of the eye and related
structures as is known in the art, such as Cystoid Macular Edema,
Asteroid Hyalosis, Pathological Myopia and Posterior Staphyloma,
Toxocariasis (Ocular Larva Migrans), Retinal Vein Occlusion,
Posterior Vitreous Detachment, Tractional Retinal Tears, Epiretinal
Membrane, Diabetic Retinopathy, Lattice Degeneration, Retinal Vein
Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g.,
age related macular degeneration such as wet AMD or dry AMD),
Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis,
Hollenhorst Plaque, Idiopathic Central Serous Chorioretinopathy,
Macular Hole, Presumed Ocular Histoplasmosis Syndrome, Retinal
Macroaneursym, Retinitis Pigmentosa, Retinal Detachment,
Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE)
Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats'
Disease, Leber's Miliary Aneurysm, Conjunctival Neoplasms, Allergic
Conjunctivitis, Vernal Conjunctivitis, Acute Bacterial
Conjunctivitis, Allergic Conjunctivitis & Vernal
Keratoconjunctivitis, Viral Conjunctivitis, Bacterial
Conjunctivitis, Chlamydial & Gonococcal Conjunctivitis,
Conjunctival Laceration, Episcleritis, Scleritis, Pingueculitis,
Pterygium, Superior Limbic Keratoconjunctivitis (SLK of Theodore),
Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane, Giant
Papillary Conjunctivitis, Terrien's Marginal Degeneration,
Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis,
Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, Bacterial
Keratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates,
Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion,
Corneal Foreign Body, Chemical Burs, Epithelial Basement Membrane
Dystrophy (EBMD), Thygeson's Superficial Punctate Keratopathy,
Corneal Laceration, Salzmann's Nodular Degeneration, Fuchs'
Endothelial Dystrophy, Crystalline Lens Subluxation, Ciliary-Block
Glaucoma, Primary Open-Angle Glaucoma, Pigment Dispersion Syndrome
and Pigmentary Glaucoma, Pseudoexfoliation Syndrome and
Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary Open Angle
Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, Pigment
Dispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure
Glaucoma, Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens
Induced Glaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative
Glaucoma, Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars
Planitis, Choroidal Rupture, Duane's Retraction Syndrome,
Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of
Cranial Nerve III, Intracranial Mass Lesions, Carotid-Cavernous
Sinus Fistula, Anterior Ischemic Optic Neuropathy, Optic Disc Edema
& Papilledema, Cranial Nerve III Palsy, Cranial Nerve IV Palsy,
Cranial Nerve VI Palsy, Cranial Nerve VII (Facial Nerve) Palsy,
Horner's Syndrome, Internuclear Ophthalmoplegia, Optic Nerve Head
Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve Head Drusen,
Demyelinating Optic Neuropathy (Optic Neuritis, Retrobulbar Optic
Neuritis), Amaurosis Fugax and Transient Ischemic Attack,
Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,
Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,
Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal Cell
Carcinoma, Herpes Zoster Ophthalmicus, Pediculosis &
Phthiriasis, Blow-out Fracture, Chronic Epiphora, Dacryocystitis,
Herpes Simplex Blepharitis, Orbital Cellulitis, Senile Entropion,
and Squamous Cell Carcinoma.
[0406] By "metabolic disease" is meant any disease or condition
affecting metabolic pathways as in known in the art. Metabolic
disease can result in an abnormal metabolic process, either
congenital due to inherited enzyme abnormality (inborn errors of
metabolism) or acquired due to disease of an endocrine organ or
failure of a metabolically important organ such as the liver. In
one embodiment, metabolic disease includes obesity, insulin
resistance, and diabetes (e.g., type I and/or type II
diabetes).
[0407] By "dermatological disease" is meant any disease or
condition of the skin, dermis, or any substructure therein such as
hair, follicle, etc. Dermatological diseases, disorders,
conditions, and traits can include psoriasis, ectopic dermatitis,
skin cancers such as melanoma and basal cell carcinoma, hair loss,
hair removal, alterations in pigmentation, and any other disease,
condition, or trait associated with the skin, dermis, or structures
therein.
[0408] By "auditory disease" is meant any disease or condition of
the auditory system, including the ear, such as the inner ear,
middle ear, outer ear, auditory nerve, and any substructures
therein. Auditory diseases, disorders, conditions, and traits can
include hearing loss, deafness, tinnitus, Meniere's Disease,
vertigo, balance and motion disorders, and any other disease,
condition, or trait associated with the ear, or structures
therein.
[0409] In one embodiment of the present invention, each sequence of
a siNA molecule of the invention is independently about 15 to about
30 nucleotides in length, in specific embodiments about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides
in length. In another embodiment, the siNA duplexes of the
invention independently comprise about 15 to about 30 base pairs
(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30). In another embodiment, one or more strands of the
siNA molecule of the invention independently comprises about 15 to
about 30 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30) that are complementary to a
target nucleic acid molecule. In yet another embodiment, siNA
molecules of the invention comprising hairpin or circular
structures are about 35 to about 55 (e.g., about 35, 40, 45, 50 or
55) nucleotides in length, or about 38 to about 44 (e.g., about 38,
39, 40, 41, 42, 43, or 44) nucleotides in length and comprising
about 15 to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25) base pairs.
[0410] As used herein "cell" is used in its usual biological sense,
and does not refer to an entire multicellular organism, e.g.,
specifically does not refer to a human. The cell can be present in
an organism, e.g., birds, plants and mammals such as humans, cows,
sheep, apes, monkeys, swine, dogs, and cats. The cell can be
prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian
or plant cell). The cell can be of somatic or germ line origin,
totipotent or pluripotent, dividing or non-dividing. The cell can
also be derived from or can comprise a gamete or embryo, a stem
cell, or a fully differentiated cell.
[0411] In one embodiment, a formulated molecular composition or
formulated siNA composition of the invention is locally
administered to relevant tissues ex vivo, or in vivo through direct
injection, catheterization, or stenting (e.g., portal vein
catherization/stenting).
[0412] In one embodiment, a formulated molecular composition or
formulated siNA composition of the invention is systemically
delivered to a subject or organism through parental administration
as is known in the art, such as via intravenous, intramuscular, or
subcutaneous injection.
[0413] In another aspect, the invention provides mammalian cells
containing one or more formulated molecular composition or
formulated siNA compositions of this invention. The one or more
formulated molecular composition or formulated siNA compositions
can independently be targeted to the same or different sites.
[0414] By "RNA" is meant a molecule comprising at least one
ribonucleotide residue. By "ribonucleotide" is meant a nucleotide
with a hydroxyl group at the 2' position of a .beta.-D-ribofuranose
moiety. The terms include double-stranded RNA, single-stranded RNA,
isolated RNA such as partially purified RNA, essentially pure RNA,
synthetic RNA, recombinantly produced RNA, as well as altered RNA
that differs from naturally occurring RNA by the addition,
deletion, substitution and/or alteration of one or more
nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of the siNA or
internally, for example at one or more nucleotides of the RNA.
Nucleotides in the RNA molecules of the instant invention can also
comprise non-standard nucleotides, such as non-naturally occurring
nucleotides or chemically synthesized nucleotides or
deoxynucleotides. These altered RNAs can be referred to as analogs
or analogs of naturally-occurring RNA.
[0415] By "subject" is meant an organism, which is a donor or
recipient of explanted cells or the cells themselves. "Subject"
also refers to an organism to which the nucleic acid molecules of
the invention can be administered. A subject can be a mammal or
mammalian cells, including a human or human cells.
[0416] The term "phosphorothioate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W
comprise a sulfur atom. Hence, the term phosphorothioate refers to
both phosphorothioate and phosphorodithioate internucleotide
linkages.
[0417] The term "phosphonoacetate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W
comprise an acetyl or protected acetyl group.
[0418] The term "thiophosphonoacetate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z comprises an
acetyl or protected acetyl group and W comprises a sulfur atom or
alternately W comprises an acetyl or protected acetyl group and Z
comprises a sulfur atom.
[0419] The term "universal base" as used herein refers to
nucleotide base analogs that form base pairs with each of the
natural DNA/RNA bases with little discrimination between them.
Non-limiting examples of universal bases include C-phenyl,
C-naphthyl and other aromatic derivatives, inosine, azole
carboxamides, and nitroazole derivatives such as 3-nitropyrrole,
4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art
(see for example Loakes, 2001, Nucleic Acids Research, 29,
2437-2447).
[0420] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of
the ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.
[0421] In a further embodiment, the formulated molecular
compositions and formulated siNA compositions can be used in
combination with other known treatments to inhibit, reduce, or
prevent diseases, traits, and conditions described herein or
otherwise known in the art in a subject or organism. For example,
the described molecules could be used in combination with one or
more known compounds, treatments, or procedures to inhibit, reduce,
or prevent diseases, traits, and conditions described herein or
otherwise known in the art in a subject or organism. In a
non-limiting example, formulated molecular composition and
formulated siNA compositions that are used to treat HCV infection
and comorbid conditions that are associated with HBV infection are
used in combination with other HCV treatments, such as HCV
vaccines; anti-HCV antibodies such as HepeX-C and Civacir; protease
inhibitors such as VX-950; pegylated interferons such as
PEG-Intron, and/or other antivirals such as Ribavirin and/or
Valopicitabine.
[0422] In one embodiment, a formulated siNA composition of the
invention comprises an expression vector comprising a nucleic acid
sequence encoding at least one polynucleotide molecule of the
invention (e.g., siNA, miRNA, RNAi inhibitor, antisense, aptamer,
decoy, ribozyme, 2-5A, triplex forming oligonucleotide, or other
nucleic acid molecule) in a manner which allows expression of the
siNA molecule. For example, the vector can contain sequence(s)
encoding both strands of a siNA molecule comprising a duplex. The
vector can also contain sequence(s) encoding a single nucleic acid
molecule that is self-complementary and thus forms a siNA molecule.
Non-limiting examples of such expression vectors are described in
Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and
Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002,
Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature
Medicine, advance online publication doi:10.1038/nm725. In one
embodiment, an expression vector of the invention comprises a
nucleic acid sequence encoding two or more siNA molecules, which
can be the same or different.
[0423] In another aspect of the invention, polynucleotides of the
invention such as siNA molecules that interact with target RNA
molecules and down-regulate gene encoding target RNA molecules (for
example target RNA molecules referred to by Genbank Accession
numbers herein) are expressed from transcription units inserted
into DNA or RNA vectors. The recombinant vectors can be DNA
plasmids or viral vectors. Polynucleotide expressing viral vectors
can be constructed based on, but not limited to, adeno-associated
virus, retrovirus, adenovirus, or alphavirus. The recombinant
vectors capable of expressing the polynucleotide molecules can be
delivered as described herein, and persist in target cells.
Alternatively, viral vectors can be used that provide for transient
expression of polynucleotide molecules. Such vectors can be
repeatedly administered as necessary. For example, once expressed,
the siNA molecules bind and down-regulate gene function or
expression via RNA interference (RNAi). Delivery of formulated
molecular compositions expressing vectors can be systemic, such as
by intravenous or intramuscular administration, by administration
to target cells ex-planted from a subject followed by
reintroduction into the subject, or by any other means that would
allow for introduction into the desired target cell.
[0424] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0425] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0426] FIG. 1 shows non-limiting examples of cationic lipid
compounds of the invention.
[0427] FIG. 2 shows non-limiting examples of acetal linked cationic
lipid compounds of the invention.
[0428] FIG. 3 shows non-limiting examples of succinyl/acyl linked
cationic lipid compounds of the invention.
[0429] FIG. 4 shows non-limiting examples of aromatic cationic
lipid compounds of the invention.
[0430] FIG. 5 shows non-limiting examples of additional cationic
lipid compounds of the invention.
[0431] FIG. 6 shows a schematic of the components of a formulated
molecular composition.
[0432] FIG. 7 shows a schematic diagram of the lamellar structure
and inverted hexagonal structure that can be adopted by a
formulated molecular composition.
[0433] FIG. 8 shows the components of L051, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0434] FIG. 9 shows the components of L073, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0435] FIG. 10 shows the components of L069, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0436] FIG. 11 shows a graph depicting the serum stability of
formulated molecular compositions L065, F2, L051, and L073 as
determined by the relative turbidity of the formulated molecular
compositions in 50% serum measured by absorbance at 500 nm.
Formulated molecular compositions L065, L051, and L073 are stable
in serum.
[0437] FIG. 12 shows a graph depicting the pH-dependent phase
transition of formulated molecular compositions L065, F2, L051, and
L073 as determined by the relative turbidity of the formulated
molecular compositions in buffer solutions ranging from pH 3.5 to
pH 9.0 measured by absorbance at 350 nm. Formulated molecular
compositions L051 and L073 each undergo a rapid pH-dependent phase
transition at pH 5.5-pH 6.5.
[0438] FIG. 13 shows a graph depicting the pH-dependent phase
transition of formulated molecular composition L069 as determined
by the relative turbidity of the formulated molecular composition
in buffer solutions ranging from pH 3.5 to pH 9.0 measured by
absorbance at 350 nm. Formulated molecular composition L069
undergoes a rapid pH-dependent phase transition at pH 5.5-pH
6.5.
[0439] FIG. 14 shows a non-limiting example of chemical
modifications of siNA molecules of the invention.
[0440] FIG. 15 shows a non-limiting example of in vitro efficacy of
siNA nanoparticles in reducing HBsAg levels in HepG2 cells. Active
chemically modified siNA molecules were designed to target HBV site
263 RNA (siNA sequences are shown in FIG. 14). The figure shows the
level of HBsAg in cells treated with formulated active siNA L051
nanoparticles (see Table IV) compared to untreated or negative
control treated cells. A dose dependent reduction in HBsAg levels
was observed in the active siNA treated cells, while no reduction
is observed in the negative control treated cells.
[0441] FIG. 16 shows a non-limiting example of in vitro efficacy of
siNA nanoparticles in reducing HBsAg levels in HepG2 cells. Active
chemically modified siNA molecules were designed to target HBV site
263 RNA (siNA sequences are shown in FIG. 14). The figure shows the
level of HBsAg in cells treated with formulated active siNA L053
and L054 nanoparticles (see Table IV) compared to untreated or
negative control treated cells. A dose dependent reduction in HBsAg
levels was observed in the active siNA treated cells, while no
reduction is observed in the negative control treated cells.
[0442] FIG. 17 shows a non-limiting example of in vitro efficacy of
siNA nanoparticles in reducing HBsAg levels in HepG2 cells. Active
chemically modified siNA molecules were designed to target HBV site
263 RNA (siNA sequences are shown in FIG. 14). The figure shows the
level of HBsAg in cells treated with formulated molecular
composition L069 comprising active siNA (see Table IV) compared to
untreated or negative control treated cells. A dose dependent
reduction in HBsAg levels was observed in the active siNA treated
cells, while no reduction is observed in the negative control
treated cells.
[0443] FIG. 18 shows a non-limiting example of the activity of
systemically administered siNA L051 (Table IV) nanoparticles in an
HBV mouse model. A hydrodynamic tail vein injection was done
containing 0.3 .mu.g of the pWTD HBV vector. The nanoparticle
encapsulated active siNA molecules were administered at 3 mg/kg/day
for three days via standard IV injection beginning 6 days post-HDI.
Groups (N=5) of animals were sacrificed at 3 and 7 days following
the last dose, and the levels of serum HBV DNA was measured. HBV
DNA titers were determined by quantitative real-time PCR and
expressed as mean log 10 copies/ml (.+-.SEM).
[0444] FIG. 19 shows a non-limiting example of the activity of
systemically administered siNA L051 (Table IV) nanoparticles in an
HBV mouse model. A hydrodynamic tail vein injection was done
containing 0.3 .mu.g of the pWTD HBV vector. The nanoparticle
encapsulated active siNA molecules were administered at 3 mg/kg/day
for three days via standard IV injection beginning 6 days post-HDI.
Groups (N=5) of animals were sacrificed at 3 and 7 days following
the last dose, and the levels of serum HBsAg was measured. The
serum HBsAg levels were assayed by ELISA and expressed as mean log
10 pg/ml (.+-.SEM).
[0445] FIG. 20 shows a non-limiting example of formulated siNA L051
(Table IV) nanoparticle constructs targeting viral replication in a
Huh7 HCV replicon system in a dose dependent manner. Active siNA
formulatations were evaluated at 1, 5, 10, and 25 nM in comparison
to untreated cells ("untreated"), and formulated inactive siNA
scrambled control constructs at the same concentration.
[0446] FIG. 21 shows a non-limiting example of formulated siNA L053
and L054 (Table IV) nanoparticle constructs targeting viral
replication in a Huh7 HCV replicon system in a dose dependent
manner. Active siNA formulatations were evaluated at 1, 5, 10, and
25 nM in comparison to untreated cells ("untreated"), and
formulated inactive siNA scrambled control constructs at the same
concentration.
[0447] FIG. 22 shows the distribution of siNA in lung tissue of
mice following intratracheal dosing of unformulated siNA,
cholesterol-conjugated siNA, and formulated siNA (formulated
molecular compositions 18.1 and 19.1). As shown, the longest half
lives of exposure in lung tissue were observed with the siNA
formulated in molecular compositions T018.1 or T019.1.
[0448] FIG. 23A shows a non-limiting example of a synthetic scheme
used for the synthesis of 3-Dimethyl
amino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadie-
noxy)propane(CLinDMA).
[0449] FIG. 23B shows a non-limiting example of an alternative
synthetic scheme used for the synthesis of
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-(cis,cis-9,12-o-
ctadecadienoxy)propane (CLinDMA).
[0450] FIG. 23C shows a non-limiting example of a synthetic scheme
used for the synthesis of N,N-Dimethyl-3,4-dilinoleyloxybenzylamine
and N,N-Dimethyl-3,4-dioleyloxybenzylamine.
[0451] FIG. 24A shows a non-limiting example of a synthetic scheme
used for the synthesis of
1-[8'-(Cholest-5-en-3.beta.-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl--
.omega.-methyl-poly(ethylene glycol) (PEG-cholesterol) and
3,4-Ditetradecoxyylbenzyl-.omega.-methyl-poly(ethylene glycol)ether
(PEG-DMB). In the Figure, PEG is PEG2000, a polydispersion which
can typically vary from .about.1500 to .about.3000 Da represented
by the formula PEG.sub.n (i.e., where n is about 33 to about 67, or
on average .about.45).
[0452] FIG. 24B shows a non-limiting example of a synthetic scheme
used for the synthesis of
1-[8'-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbam-
oyl-.omega.-methyl-poly(ethylene glycol) (PEG-DMG). In the Figure,
PEG is PEG2000, a polydispersion which can typically vary from
.about.1500 to .about.3000 Da represented by the formula PEG.sub.n
(i.e., where n is about 33 to about 67, or on average
.about.45).
[0453] FIG. 25 shows the components of L083, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0454] FIG. 26 shows the components of L077, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0455] FIG. 27 shows the components of L080, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0456] FIG. 28 shows the components of L082, a serum-stable
formulated molecular composition that undergoes a rapid
pH-dependent phase transition.
[0457] FIG. 29 shows a non-limiting example of the activity of
systemically administered siNA L077, L069, L080, L082, L083, L060,
L061, and L051 (Table IV) nanoparticles in an HBV mouse model. A
hydrodynamic tail vein injection was done containing 0.3 .mu.g of
the pWTD HBV vector. The nanoparticle encapsulated active siNA
molecules were administered at 3 mg/kg/day for three days via
standard IV injection beginning 6 days post-HDI. Groups (N=5) of
animals were sacrificed at 3 and 7 days following the last dose,
and the levels of serum HBV DNA was measured. HBV DNA titers were
determined by quantitative real-time PCR and expressed as mean log
10 copies/ml (.+-.SEM).
[0458] FIG. 30 shows a non-limiting example of the dose response
activity of systemically administered siNA L083 and L084 (Table IV)
nanoparticles in an HBV mouse model. A hydrodynamic tail vein
injection was done containing 0.3 .mu.g of the pWTD HBV vector. The
nanoparticle encapsulated active siNA molecules were administered
at 3 mg/kg/day for three days via standard IV injection beginning 6
days post-HDI. Groups (N=5) of animals were sacrificed at 3 and 7
days following the last dose, and the levels of serum HBsAg was
measured. The serum HBsAg levels were assayed by ELISA and
expressed as mean log 10 pg/ml (.+-.SEM).
[0459] FIG. 31 shows a non-limiting example of the dose response
activity of systemically administered siNA L077 (Table IV)
nanoparticles in an HBV mouse model. A hydrodynamic tail vein
injection was done containing 0.3 .mu.g of the pWTD HBV vector. The
nanoparticle encapsulated active siNA molecules were administered
at 3 mg/kg/day for three days via standard IV injection beginning 6
days post-HDI. Groups (N=5) of animals were sacrificed at 3 and 7
days following the last dose, and the levels of serum HBsAg was
measured. The serum HBsAg levels were assayed by ELISA and
expressed as mean log 10 pg/ml (.+-.SEM).
[0460] FIG. 32 shows a non-limiting example of the dose response
activity of systemically administered siNA L080 (Table IV)
nanoparticles in an HBV mouse model. A hydrodynamic tail vein
injection was done containing 0.3 .mu.g of the pWTD HBV vector. The
nanoparticle encapsulated active siNA molecules were administered
at 3 mg/kg/day for three days via standard IV injection beginning 6
days post-HDI. Groups (N=5) of animals were sacrificed at 3 and 7
days following the last dose, and the levels of serum HBsAg was
measured. The serum HBsAg levels were assayed by ELISA and
expressed as mean log 10 pg/ml (.+-.SEM).
[0461] FIG. 33 shows a non-limiting example of the serum stability
of siNA L077, L080, L082, and L083 (Table IV) nanoparticle
formulations.
[0462] FIG. 34 shows a graph depicting the pH-dependent phase
transition of siNA L077, L080, L082, and L083 (Table IV)
nanoparticle formulations as determined by the relative turbidity
of the formulated molecular composition in buffer solutions ranging
from pH 3.5 to pH 9.0 measured by absorbance at 350 nm. Formulated
molecular composition L069 undergoes a rapid pH-dependent phase
transition at pH 5.5-pH 6.5.
[0463] FIG. 35 shows efficacy data for LNP 58 and LNP 98
formulations targeting MapK14 site 1033 in RAW 264.7 mouse
macrophage cells compared to LFK2000 and a formulated irrelevant
siNA control.
[0464] FIG. 36 shows efficacy data for LNP 98 formulations
targeting MapK14 site 1033 in MM14.Lu normal mouse lung cells
compared to LFK2000 and a formulated irrelevant siNA control.
[0465] FIG. 37 shows efficacy data for LNP 54, LNP 97, and LNP 98
formulations targeting MapK14 site 1033 in 6.12 B lymphocyte cells
compared to LFK2000 and a formulated irrelevant siNA control.
[0466] FIG. 38 shows efficacy data for LNP 98 formulations
targeting MapK14 site 1033 in NIH 3T3 cells compared to LFK2000 and
a formulated irrelevant siNA control.
[0467] FIG. 39 shows the dose-dependent reduction of MapK14 RNA via
MapK14 LNP 54 and LNP 98 formulated siNAs in RAW 264.7 cells.
[0468] FIG. 40 shows the dose-dependent reduction of MapK14 RNA via
MapK14 LNP 98 formulated siNAs in MM14.Lu cells.
[0469] FIG. 41 shows the dose-dependent reduction of MapK14 RNA via
MapK14 LNP 97 and LNP 98 formulated siNAs in 6.12 B cells.
[0470] FIG. 42 shows the dose-dependent reduction of MapK14 RNA via
MapK14 LNP 98 formulated siNAs in NIH 3T3 cells.
[0471] FIG. 43 shows a non-limiting example of reduced airway
hyper-responsiveness from treatment with LNP-51 formulated siNAs
targeting IL-4R in a mouse model of OVA challenge mediated airway
hyper-responsiveness. Active formulated siNAs were tested at 0.01,
0.1, and 1.0 mg/kg and were compared to LNP vehicle along and
untreated (naive) animals.
[0472] FIG. 44 shows a non-limiting example of LNP formulated siNA
mediated inhibition of huntingtin (htt) gene expression in vivo.
Using Alzet osmotic pumps, siNAs encapsulated in LNPs were infused
into mouse lateral ventrical or striatum for 7 or 14 days,
respectively, at concentrations ranging from 0.1 to 1 mg/ml (total
dose ranging from 8.4 to 84 .mu.g). Animals treated with active
siNA formulated with LNP-098 or LNP-061 were compared to mismatch
control siNA formulated with LNP-061 and untreated animal controls.
Huntingtin (htt) gene expression levels were determined by
QPCR.
DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention
[0473] Aptamer: Nucleic acid aptamers can be selected to
specifically bind to a particular ligand of interest (see for
example Gold et al., U.S. Pat. No. 5,567,588 and U.S. Pat. No.
5,475,096, Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody
and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol.
Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and
Patel, 2000, Science, 287, 820; and Jayasena, 1999, Clinical
Chemistry, 45, 1628). For example, the use of in vitro selection
can be applied to evolve nucleic acid aptamers with binding
specificity for CylA. Nucleic acid aptamers can include chemical
modifications and linkers as described herein. Nucleic apatmers of
the invention can be double stranded or single stranded and can
comprise one distinct nucleic acid sequence or more than one
nucleic acid sequences complexed with one another. Aptamer
molecules of the invention that bind to CylA, can modulate the
protease activity of CylA and subsequent activation of cytolysin,
and therefore modulate the acute toxicity accociated with
enterococcal infection.
[0474] Antisense: Antisense molecules can be modified or unmodified
RNA, DNA, or mixed polymer oligonucleotides and primarily function
by specifically binding to matching sequences resulting in
modulation of peptide synthesis (Wu-Pong, November 1994, BioPharm,
20-33). The antisense oligonucleotide binds to target RNA by Watson
Crick base-pairing and blocks gene expression by preventing
ribosomal translation of the bound sequences either by steric
blocking or by activating RNase H enzyme. Antisense molecules may
also alter protein synthesis by interfering with RNA processing or
transport from the nucleus into the cytoplasm (Mukhopadhyay &
Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
[0475] In addition, binding of single stranded DNA to RNA may
result in nuclease degradation of the heteroduplex (Wu-Pong, supra;
Crooke, supra). To date, the only backbone modified DNA chemistry
which will act as substrates for RNase H are phosphorothioates,
phosphorodithioates, and borontrifluoridates. Recently, it has been
reported that 2'-arabino and 2'-fluoro arabino-containing oligos
can also activate RNase H activity.
[0476] A number of antisense molecules have been described that
utilize novel configurations of chemically modified nucleotides,
secondary structure, and/or RNase H substrate domains (Woolf et
al., U.S. Pat. No. 5,989,912; Thompson et al., U.S. Ser. No.
60/082,404 which was filed on Apr. 20, 1998; Hartmann et al., U.S.
Ser. No. 60/101,174 which was filed on Sep. 21, 1998) all of these
are incorporated by reference herein in their entirety.
[0477] Antisense DNA can be used to target RNA by means of DNA-RNA
interactions, thereby activating RNase H, which digests the target
RNA in the duplex. Antisense DNA can be chemically synthesized or
can be expressed via the use of a single stranded DNA intracellular
expression vector or the equivalent thereof.
[0478] Triplex Forming Oligonucleotides (TFO): Single stranded
oligonucleotide can be designed to bind to genomic DNA in a
sequence specific manner. TFOs can be comprised of pyrimidine-rich
oligonucleotides which bind DNA helices through Hoogsteen
Base-pairing (Wu-Pong, supra). In addition, TFOs can be chemically
modified to increase binding affinity to target DNA sequences. The
resulting triple helix composed of the DNA sense, DNA antisense,
and TFO disrupts RNA synthesis by RNA polymerase. The TFO mechanism
can result in gene expression or cell death since binding may be
irreversible (Mukhopadhyay & Roth, supra)
[0479] 2'-5' Oligoadenylates: The 2-5A system is an
interferon-mediated mechanism for RNA degradation found in higher
vertebrates (Mitra et al., 1996, Proc Nat Acad Sci USA 93,
6780-6785). Two types of enzymes, 2-5A synthetase and RNase L, are
required for RNA cleavage. The 2-5A synthetases require double
stranded RNA to form 2'-5' oligoadenylates (2-5A). 2-5A then acts
as an allosteric effector for utilizing RNase L, which has the
ability to cleave single stranded RNA. The ability to form 2-5A
structures with double stranded RNA makes this system particularly
useful for modulation of viral replication.
[0480] (2'-5') oligoadenylate structures can be covalently linked
to antisense molecules to form chimeric oligonucleotides capable of
RNA cleavage (Torrence, supra). These molecules putatively bind and
activate a 2-5A-dependent RNase, the oligonucleotide/enzyme complex
then binds to a target RNA molecule which can then be cleaved by
the RNase enzyme. The covalent attachment of 2'-5' oligoadenylate
structures is not limited to antisense applications, and can be
further elaborated to include attachment to nucleic acid molecules
of the instant invention.
[0481] Enzymatic Nucleic Acid: Several varieties of naturally
occurring enzymatic RNAs are presently known (Doherty and Doudna,
2001, Annu. Rev. Biophys. Biomol. Struct., 30, 457-475; Symons,
1994, Curr. Opin. Struct. Biol., 4, 322-30). In addition, several
in vitro selection (evolution) strategies (Orgel, 1979, Proc. R.
Soc. London, B 205, 435) have been used to evolve new nucleic acid
catalysts capable of catalyzing cleavage and ligation of
phosphodiester linkages (Joyce, 1989, Gene, 82, 83-87; Beaudry et
al., 1992, Science 257, 635-641; Joyce, 1992, Scientific American
267, 90-97; Breaker et al., 1994, TIBTECH 12, 268; Bartel et al.,
1993, Science 261:1411-1418; Szostak, 1993, TIBS 17, 89-93; Kumar
et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op. Biotech.,
7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94, 4262;
Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994,
supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995,
supra; Vaish et al., 1997, Biochemistry 36, 6495). Each can
catalyze a series of reactions including the hydrolysis of
phosphodiester bonds in trans (and thus can cleave other RNA
molecules) under physiological conditions.
[0482] The enzymatic nature of an enzymatic nucleic acid has
significant advantages, such as the concentration of nucleic acid
necessary to affect a therapeutic treatment is low. This advantage
reflects the ability of the enzymatic nucleic acid molecule to act
enzymatically. Thus, a single enzymatic nucleic acid molecule is
able to cleave many molecules of target RNA. In addition, the
enzymatic nucleic acid molecule is a highly specific modulator,
with the specificity of modulation depending not only on the
base-pairing mechanism of binding to the target RNA, but also on
the mechanism of target RNA cleavage. Single mismatches, or
base-substitutions, near the site of cleavage can be chosen to
completely eliminate catalytic activity of an enzymatic nucleic
acid molecule.
[0483] Nucleic acid molecules having an endonuclease enzymatic
activity are able to repeatedly cleave other separate RNA molecules
in a nucleotide base sequence-specific manner. With proper design
and construction, such enzymatic nucleic acid molecules can be
targeted to any RNA transcript, and efficient cleavage achieved in
vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck, 1987 Nature
328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987;
Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff and
Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and
Jefferies et al., 17 Nucleic Acids Research 1371, 1989; Chartrand
et al., 1995, Nucleic Acids Research 23, 4092; Santoro et al.,
1997, PNAS 94, 4262).
[0484] Because of their sequence specificity, trans-cleaving
enzymatic nucleic acid molecules show promise as therapeutic agents
for human disease (Usman & McSwiggen, 1995 Ann. Rep. Med. Chem.
30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38,
2023-2037). Enzymatic nucleic acid molecule can be designed to
cleave specific RNA targets within the background of cellular RNA.
Such a cleavage event renders the RNA non-functional and abrogates
protein expression from that RNA. In this manner, synthesis of a
protein associated with a disease state can be selectively
modulated (Warashina et al., 1999, Chemistry and Biology, 6,
237-250).
[0485] The present invention also features nucleic acid sensor
molecules or allozymes having sensor domains comprising nucleic
acid decoys and/or aptamers of the invention. Interaction of the
nucleic acid sensor molecule's sensor domain with a molecular
target can activate or inactivate the enzymatic nucleic acid domain
of the nucleic acid sensor molecule, such that the activity of the
nucleic acid sensor molecule is modulated in the presence of the
target-signaling molecule. The nucleic acid sensor molecule can be
designed to be active in the presence of the target molecule or
alternately, can be designed to be inactive in the presence of the
molecular target. For example, a nucleic acid sensor molecule is
designed with a sensor domain comprising an aptamer with binding
specificity for a ligand. In a non-limiting example, interaction of
the ligand with the sensor domain of the nucleic acid sensor
molecule can activate the enzymatic nucleic acid domain of the
nucleic acid sensor molecule such that the sensor molecule
catalyzes a reaction, for example cleavage of RNA that encodes the
ligand. In this example, the nucleic acid sensor molecule is
activated in the presence of ligand, and can be used as a
therapeutic to treat a disease or codition associated with the
ligand. Alternately, the reaction can comprise cleavage or ligation
of a labeled nucleic acid reporter molecule, providing a useful
diagnostic reagent to detect the presence of ligand in a
system.
[0486] RNA interference: The discussion that follows discusses the
proposed mechanism of RNA interference mediated by short
interfering RNA as is presently known, and is not meant to be
limiting and is not an admission of prior art. Applicant
demonstrates herein that chemically-modified short interfering
nucleic acids possess similar or improved capacity to mediate RNAi
as do siRNA molecules and are expected to possess improved
stability and activity in vivo; therefore, this discussion is not
meant to be limiting only to siRNA and can be applied to siNA as a
whole. By "improved capacity to mediate RNAi" or "improved RNAi
activity" is meant to include RNAi activity measured in vitro
and/or in vivo where the RNAi activity is a reflection of both the
ability of the siNA to mediate RNAi and the stability of the siNAs
of the invention. In this invention, the product of these
activities can be increased in vitro and/or in vivo compared to an
all RNA siRNA or a siNA containing a plurality of ribonucleotides.
In some cases, the activity or stability of the siNA molecule can
be decreased (i.e., less than ten-fold), but the overall activity
of the siNA molecule is enhanced in vitro and/or in vivo.
[0487] RNA interference refers to the process of sequence specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806).
The corresponding process in plants is commonly referred to as
post-transcriptional gene silencing or RNA silencing and is also
referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent
the expression of foreign genes which is commonly shared by diverse
flora and phyla (Fire et al., 1999, Trends Genet., 15, 358). Such
protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived
from viral infection or the random integration of transposon
elements into a host genome via a cellular response that
specifically destroys homologous single-stranded RNA or viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi
response though a mechanism that has yet to be fully characterized.
This mechanism appears to be different from the interferon response
that results from dsRNA-mediated activation of protein kinase PKR
and 2',5'-oligoadenylate synthetase resulting in non-specific
cleavage of mRNA by ribonuclease L.
[0488] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as Dicer. Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNAs) (Berstein et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from Dicer
activity are typically about 21 to about 23 nucleotides in length
and comprise about 19 base pair duplexes. Dicer has also been
implicated in the excision of 21- and 22-nucleotide small temporal
RNAs (stRNAs) from precursor RNA of conserved structure that are
implicated in translational control (Hutvagner et al., 2001,
Science, 293, 834). The RNAi response also features an endonuclease
complex containing a siRNA, commonly referred to as an RNA-induced
silencing complex (RISC), which mediates cleavage of
single-stranded RNA having sequence homologous to the siRNA.
Cleavage of the target RNA takes place in the middle of the region
complementary to the guide sequence of the siRNA duplex (Elbashir
et al., 2001, Genes Dev., 15, 188). In addition, RNA interference
can also involve small RNA (e.g., micro-RNA or miRNA) mediated gene
silencing, presumably though cellular mechanisms that regulate
chromatin structure and thereby prevent transcription of target
gene sequences (see for example Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237). As such, siNA molecules of the invention can be used to
mediate gene silencing via interaction with RNA transcripts or
alternately by interaction with particular gene sequences, wherein
such interaction results in gene silencing either at the
transcriptional level or post-transcriptional level.
[0489] RNAi has been studied in a variety of systems. Fire et al.,
1998, Nature, 391, 806, were the first to observe RNAi in C.
elegans. Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe
RNAi mediated by dsRNA in mouse embryos. Hammond et al., 2000,
Nature, 404, 293, describe RNAi in Drosophila cells transfected
with dsRNA. Elbashir et al., 2001, Nature, 411, 494, describe RNAi
induced by introduction of duplexes of synthetic 21-nucleotide RNAs
in cultured mammalian cells including human embryonic kidney and
HeLa cells. Recent work in Drosophila embryonic lysates has
revealed certain requirements for siRNA length, structure, chemical
composition, and sequence that are essential to mediate efficient
RNAi activity. These studies have shown that 21 nucleotide siRNA
duplexes are most active when containing two 2-nucleotide
3'-terminal nucleotide overhangs. Furthermore, substitution of one
or both siRNA strands with 2'-deoxy or 2'-O-methyl nucleotides
abolishes RNAi activity, whereas substitution of 3'-terminal siRNA
nucleotides with deoxy nucleotides was shown to be tolerated.
Mismatch sequences in the center of the siRNA duplex were also
shown to abolish RNAi activity. In addition, these studies also
indicate that the position of the cleavage site in the target RNA
is defined by the 5'-end of the siRNA guide sequence rather than
the 3'-end (Elbashir et al., 2001, EMBO J. , 20, 6877). Other
studies have indicated that a 5'-phosphate on the
target-complementary strand of a siRNA duplex is required for siRNA
activity and that ATP is utilized to maintain the 5'-phosphate
moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309);
however, siRNA molecules lacking a 5'-phosphate are active when
introduced exogenously, suggesting that 5'-phosphorylation of siRNA
constructs may occur in vivo.
Synthesis of Nucleic Acid Molecules
[0490] Synthesis of nucleic acids greater than 100 nucleotides in
length is difficult using automated methods and the therapeutic
cost of such molecules is prohibitive. In this invention, small
nucleic acid motifs ("small" refers to nucleic acid motifs no more
than 100 nucleotides in length, preferably no more than 80
nucleotides in length, and most preferably no more than 50
nucleotides in length; e.g., individual siNA oligonucleotide
sequences or siNA sequences synthesized in tandem) are preferably
used for exogenous delivery. The simple structure of these
molecules increases the ability of the nucleic acid to invade
targeted regions of protein and/or RNA structure. Exemplary
molecules of the instant invention are chemically synthesized, and
others can similarly be synthesized.
[0491] Oligonucleotides (e.g., certain modified oligonucleotides or
portions of oligonucleotides lacking ribonucleotides) are
synthesized using protocols known in the art, for example as
described in Caruthers et al., 1992, Methods in Enzymology 211,
3-19, Thompson et al., International PCT Publication No. WO
99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684,
Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al.,
1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.
6,001,311. All of these references are incorporated herein by
reference. 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.
In a non-limiting example, small scale syntheses are conducted on a
394 Applied Biosystems, Inc. synthesizer using a 0.2 .mu.mol scale
protocol with a 2.5 min coupling step for 2'-O-methylated
nucleotides and a 45 second coupling step for 2'-deoxy nucleotides
or 2'-deoxy-2'-fluoro nucleotides. Table II outlines the amounts
and the contact times of the reagents used in the synthesis cycle.
Alternatively, syntheses at the 0.2 .mu.mol scale can be performed
on a 96-well plate synthesizer, such as the instrument produced by
Protogene (Palo Alto, Calif.) with minimal modification to the
cycle. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of
2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl
tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each
coupling cycle of 2'-O-methyl residues relative to polymer-bound
5'-hydroxyl. A 22-fold excess (40 .mu.L of 0.11 M=4.4 .mu.mol) of
deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40
.mu.L of 0.25 M=10 .mu.mol) can be used in each coupling cycle of
deoxy residues relative to polymer-bound 5'-hydroxyl. Average
coupling yields on the 394 Applied Biosystems, Inc. synthesizer,
determined by colorimetric quantitation of the trityl fractions,
are typically 97.5-99%. Other oligonucleotide synthesis reagents
for the 394 Applied Biosystems, Inc. synthesizer include the
following: detritylation solution is 3% TCA in methylene chloride
(ABI); capping is performed with 16% N-methyl imidazole in THF
(ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and
oxidation solution is 16.9 mM I.sub.2, 49 mM pyridine, 9% water in
THF (PerSeptive Biosystems, Inc.). Burdick & Jackson Synthesis
Grade acetonitrile is used directly from the reagent bottle.
S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from
the solid obtained from American International Chemical, Inc.
Alternately, for the introduction of phosphorothioate linkages,
Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in
acetonitrile) is used.
[0492] Deprotection of the DNA-based oligonucleotides is performed
as follows: the polymer-bound trityl-on oligoribonucleotide is
transferred to a 4 mL glass screw top vial and suspended in a
solution of 40% aqueous methylamine (1 mL) at 65.degree. C. for 10
minutes. After cooling to -20.degree. C., the supernatant is
removed from the polymer support. The support is washed three times
with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is
then added to the first supernatant. The combined supernatants,
containing the oligoribonucleotide, are dried to a white
powder.
[0493] The method of synthesis used for RNA including certain siNA
molecules of the invention follows the procedure as described in
Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al.,
1990, Nucleic Acids Res., 18, 5433; and Wincott et al., 1995,
Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol.
Bio., 74, 59, and makes use of common nucleic acid protecting and
coupling groups, such as dimethoxytrityl at the 5'-end, and
phosphoramidites at the 3'-end. In a non-limiting example, small
scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 .mu.mol scale protocol with a 7.5 min
coupling step for alkylsilyl protected nucleotides and a 2.5 min
coupling step for 2'-O-methylated nucleotides. Table II outlines
the amounts and the contact times of the reagents used in the
synthesis cycle. Alternatively, syntheses at the 0.2 .mu.mol scale
can be done on a 96-well plate synthesizer, such as the instrument
produced by Protogene (Palo Alto, Calif.) with minimal modification
to the cycle. A 33-fold excess (60 .mu.L of 0.11M=6.6 .mu.mol of
2'-O-methyl phosphoramidite and a 75-fold excess of S-ethyl
tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each
coupling cycle of 2'-O-methyl residues relative to polymer-bound
5'-hydroxyl. A 66-fold excess (120 .mu.L of 0.11 M=13.2 .mu.mol) of
alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess
of S-ethyl tetrazole (120 .mu.L of 0.25 M=30 .mu.mol) can be used
in each coupling cycle of ribo residues relative to polymer-bound
5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems,
Inc. synthesizer, determined by colorimetric quantitation of the
trityl fractions, are typically 97.5-99%. Other oligonucleotide
synthesis reagents for the 394 Applied Biosystems, Inc. synthesizer
include the following: detritylation solution is 3% TCA in
methylene chloride (ABI); capping is performed with 16% N-methyl
imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in
THF (ABI); oxidation solution is 16.9 mM I.sub.2, 49 mM pyridine,
9% water in THF (PerSeptive Biosystems, Inc.). Burdick &
Jackson Synthesis Grade acetonitrile is used directly from the
reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile)
is made up from the solid obtained from American International
Chemical, Inc. Alternately, for the introduction of
phosphorothioate linkages, Beaucage reagent
(3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is
used.
[0494] Deprotection of the RNA is performed using either a two-pot
or one-pot protocol.
[0495] For the two-pot protocol, the polymer-bound trityl-on
oligoribonucleotide is transferred to a 4 mL glass screw top vial
and suspended in a solution of 40% aq. methylamine (1 mL) at
65.degree. C. for 10 min. After cooling to -20.degree. C., the
supernatant is removed from the polymer support. The support is
washed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and
the supernatant is then added to the first supernatant. The
combined supernatants, containing the oligoribonucleotide, are
dried to a white powder. The base deprotected oligoribonucleotide
is resuspended in anhydrous TEA/HF/NMP solution (300 .mu.L it of a
solution of 1.5 mL N-methylpyrrolidinone, 750 .mu.L TEA and 1 mL
TEA.cndot.3HF to provide a 1.4 M HF concentration) and heated to
65.degree. C. After 1.5 h, the oligomer is quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0496] Alternatively, for the one-pot protocol, the polymer-bound
trityl-on oligoribonucleotide is transferred to a 4 mL glass screw
top vial and suspended in a solution of 33% ethanolic
methylamine/DMSO: 1/1 (0.8 mL) at 65.degree. C. for 15 minutes. The
vial is brought to room temperature TEA.cndot.3HF (0.1 mL) is added
and the vial is heated at 65.degree. C. for 15 minutes. The sample
is cooled at -20.degree. C. and then quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0497] For purification of the trityl-on oligomers, the quenched
NH.sub.4HCO.sub.3 solution is loaded onto a C-18 containing
cartridge that had been prewashed with acetonitrile followed by 50
mM TEAA. After washing the loaded cartridge with water, the RNA is
detritylated with 0.5% TFA for 13 minutes. The cartridge is then
washed again with water, salt exchanged with 1 M NaCl and washed
with water again. The oligonucleotide is then eluted with 30%
acetonitrile.
[0498] The average stepwise coupling yields are typically >98%
(Wincott et al., 1995 Nucleic Acids Res. 23, 2677-2684). Those of
ordinary skill in the art will recognize that the scale of
synthesis can be adapted to be larger or smaller than the example
described above including but not limited to 96-well format.
[0499] Alternatively, the nucleic acid molecules of the present
invention can be synthesized separately and joined together
post-synthetically, for example, by ligation (Moore et al., 1992,
Science 256, 9923; Draper et al., International PCT publication No.
WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19,
4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951;
Bellon et al., 1997, Bioconjugate Chem. 8, 204), or by
hybridization following synthesis and/or deprotection.
[0500] The siNA molecules of the invention can also be synthesized
via a tandem synthesis methodology as described in Example 1
herein, wherein both siNA strands are synthesized as a single
contiguous oligonucleotide fragment or strand separated by a
cleavable linker which is subsequently cleaved to provide separate
siNA fragments or strands that hybridize and permit purification of
the siNA duplex. The linker can be a polynucleotide linker or a
non-nucleotide linker. The tandem synthesis of siNA as described
herein can be readily adapted to both multiwell/multiplate
synthesis platforms such as 96 well or similarly larger multi-well
platforms. The tandem synthesis of siNA as described herein can
also be readily adapted to large scale synthesis platforms
employing batch reactors, synthesis columns and the like.
[0501] A siNA molecule can also be assembled from two distinct
nucleic acid strands or fragments wherein one fragment includes the
sense region and the second fragment includes the antisense region
of the RNA molecule.
[0502] The nucleic acid molecules of the present invention can be
modified extensively to enhance stability by modification with
nuclease resistant groups, for example, 2'-amino, 2'-C-allyl,
2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren,
1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31,
163). siNA constructs can be purified by gel electrophoresis using
general methods or can be purified by high pressure liquid
chromatography (HPLC; see Wincott et al., supra, the totality of
which is hereby incorporated herein by reference) and re-suspended
in water.
[0503] In another aspect of the invention, siNA molecules of the
invention are expressed from transcription units inserted into DNA
or RNA vectors. The recombinant vectors can be DNA plasmids or
viral vectors. siNA expressing viral vectors can be constructed
based on, but not limited to, adeno-associated virus, retrovirus,
adenovirus, or alphavirus. The recombinant vectors capable of
expressing the siNA molecules can be delivered as described herein,
and persist in target cells. Alternatively, viral vectors can be
used that provide for transient expression of siNA molecules.
Preparation of Lipid Nanoparticle (LNP) Compositions
[0504] In one embodiment, the invention features a process for
producing a lipid nanoparticle composition of the invention. The
process typically includes providing an aqueous solution comprising
a biologically active molecule of the invention (e.g., a siNA,
miRNA, siRNA, or RNAi inhibitor), in a first reservoir, the first
reservoir in fluid communication with an organic lipid solution in
a second reservoir, and mixing the aqueous solution with the
organic lipid solution, followed by an incubation step, a
diafiltration step, and a final concentration step. In one
embodiment, the aqueous solution such as a buffer, comprises a
biologically active molecule, such that the biologically active
molecule is encapsulated in the lipid nanoparticle as a result of
the process.
[0505] In another embodiment, the invention features apparatus for
producing a lipid nanoparticle (LNP) composition including a
biologically active molecule. The apparatus typically includes a
first reservoir for holding an aqueous solution, and a second
reservoir for holding an organic lipid solution, wherein the
aqueous solution solution includes the biologically active
molecule. The apparatus also typically includes a pump mechanism
configured to pump the aqueous and the organic lipid solutions into
a mixing region or mixing chamber at substantially equal flow
rates. In one embodiment, the mixing region or mixing chamber
comprises a T coupling or equivalent thereof, which allows the
aqueous and organic fluid streams to combine as input into the T
connector and the resulting combined aqueous and organic solutions
to exit out of the T connector into a collection reservoir or
equivalent thereof. In operation, the organic lipid solution mixes
with the aqueous solution in the mixing region to form a desired
lipid nanoparticle composition after incubation, difiltration, and
concentration.
[0506] In one embodiment, the invention features a process for
synthesizing a lipid nanoparticle composition of the invention
comprising: (a) providing an aqueous solution comprising a
biologically active molecule of the invention (e.g., a siNA, miRNA,
siRNA, or RNAi inhibitor); (b) providing an organic solution
comprising LNP components of the invention (see for example LNP
components shown in Table IV); (c) mixing the aqueous solution with
the organic solution; (d) incubating the resulting combined aqueous
and organic solution prior to (e) diluiton; (f) ultrafiltration;
and (g) final concentration under conditions suitable to produce
the lipid nanoparticle composition. In one embodiment, the
biologically active molecule is encapsulated in the lipid
nanoparticle as a result of the process.
[0507] In one embodiment, the present invention provides a method
for the preparation of a lipid nanoparticle (LNP) composition
comprising a biologically active molecule, comprising: (a)
preparing a solution of the biologically active molecule(s) of
interest (e.g., siNA, miRNA, RNAi inhibitor) in a suitable buffer;
(b) preparing a solution of lipid components (e.g. CLinDMA, DSPC,
Cholesterol, PEG-DMG), and/or Linoleyl alcohol) in a suitable
buffer; (c) mixing the lipid component solution and the
biologically active molecule solution together under conditions
suitable for particle formation; (d) incubating the resulting
mixture prior to (e) dilution with a suitable buffer; (0
ultrafiltration; and (g) final concentration of the LNP composition
(see for example Table VI and Example 17 herein).
[0508] In one embodiment, the buffer of (a) is an aqueous buffer
such as a citrate buffer. In another embodiment, the buffer of (b)
comprises an organic alcohol such as ethanol. In one embodiment,
the mixing in (c) comprises utilizing a pumping apparatus that
combines a first fluid stream of the solution of (a) and a second
fluid stream of the solution of (b) into a mixing region at
substantially equal flow rates to form the lipid nanoparticle
composition. In another embodiment, the incubation of (d) comprises
allowing the resulting in-process solution of (c) to stand in a
vessel for about 12 to about 100 hours (preferably about 12 to
about 24 hours) at about room temperature and optionally protected
from light. In one embodiment, the dilution (e) involves dilution
with aqueous buffer (e.g., citrate buffer) using a pump system
(such as a diaphragm pump). In one embodiment, ultrafiltration (f)
comprises concentration of the diluted LNP solution followed by
diafiltration, for example using a suitable pumping system (e.g.,
pumping apparatus such as a Quatroflow pump or equivalent thereof)
in conjuction with a suitable ultrafiltration membrane (e.g., GE NP
UFP-100-C-35A or equivalent thereof).
[0509] In another group of embodiments, the present invention
provides a method for the preparation of a lipid nanoparticle
(LNP), comprising: (a) preparing a mixture comprising cationic
lipids and noncationic lipids in an organic solvent; (b) contacting
an aqueous solution of molecule of interest with the mixture in
step (a) to provide a clear single phase; and (c) removing the
organic solvent to provide a suspension of molecule-lipid
particles, wherein the molecule of interest is encapsulated in a
lipid bilayer, and the particles are stable in serum and have a
size of from about 50 to about 150 nm or alternately 50 to about
600 nm.
[0510] The selection of an organic solvent will typically involve
consideration of solvent polarity and the ease with which the
solvent can be removed at the later stages of particle formation.
The organic solvent, which is also used as a solubilizing agent, is
in an amount sufficient to provide a clear single phase mixture of
biologically active molecules and lipids. Suitable solvents
include, but are not limited to, chloroform, dichloromethane,
diethylether, cyclohexane, cyclopentane, benzene, toluene,
methanol, or other aliphatic alcohols such as propanol,
isopropanol, butanol, tert-butanol, iso-butanol, pentanol and
hexanol. Combinations of two or more solvents can also be used in
the present invention.
[0511] Contacting the molecules of interest with the organic
solution of cationic and neutral lipids is accomplished by mixing
together a first solution of the molecule of interest, which is
typically an aqueous solution, and a second organic solution of the
lipids. One of skill in the art will understand that this mixing
can take place by any number of methods, for example by mechanical
means such as by using vortex mixers.
[0512] After the molecule of interest has been contacted with the
organic solution of lipids, the organic solvent is removed, thus
forming an aqueous suspension of serum-stable molecule-lipid
particles. The methods used to remove the organic solvent will
typically involve evaporation at reduced pressures or blowing a
stream of inert gas (e.g., nitrogen or argon) across the
mixture.
[0513] The formulated molecular compositions thus formed will
typically be sized from about 50 nm to 150 nm or alternately from
about 50 nm to 600 nm. To achieve further size reduction or
homogeneity of size in the particles, sizing can be conducted as
described above.
[0514] In other embodiments, the methods will further comprise
adding nonlipid polycations which are useful to effect the
transformation of cells using the present compositions. Examples of
suitable nonlipid polycations include, but are limited to,
hexadimethrine bromide (sold under the brandname 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.
[0515] In certain embodiments, the formation of the lipid
nanoparticle (LNP) compositions can be carried out either in a
mono-phase system (e.g., a Bligh and Dyer monophase or similar
mixture of aqueous and organic solvents) or in a two-phase system
with suitable mixing.
[0516] When formation of the lipid nanoparticle (LNP) is carried
out in a mono-phase system, the cationic lipids and molecules of
interest are each dissolved in a volume of the mono-phase mixture.
Combination of the two solutions provides a single mixture in which
the complexes form. Alternatively, the complexes can form in
two-phase mixtures in which the cationic lipids bind to the
molecule (which is present in the aqueous phase), and "pull" it
into the organic phase.
[0517] In another embodiment, the present invention provides a
method for the preparation of lipid nanoparticle (LNP)
compositions, comprising: (a) contacting molecules of interest with
a solution comprising noncationic lipids and a detergent to form a
molecule-lipid mixture; (b) contacting cationic lipids with the
molecule-lipid mixture to neutralize a portion of the negative
charge of the molecule of interest and form a charge-neutralized
mixture of molecules and lipids; and (c) removing the detergent
from the charge-neutralized mixture to provide the lipid
nanoparticle (LNP) composition.
[0518] In one group of embodiments, the solution of neutral lipids
and detergent is an aqueous solution. Contacting the molecules of
interest with the solution of neutral lipids and detergent is
typically accomplished by mixing together a first solution of the
molecule of interst and a second solution of the lipids and
detergent. One of skill in the art will understand that this mixing
can take place by any number of methods, for example, by mechanical
means such as by using vortex mixers. Preferably, the molecule
solution is also a detergent solution. The amount of neutral lipid
which is used in the present method is typically determined based
on the amount of cationic lipid used, and is typically of from
about 0.2 to 5 times the amount of cationic lipid, preferably from
about 0.5 to about 2 times the amount of cationic lipid used.
[0519] The molecule-lipid mixture thus formed is contacted with
cationic lipids to neutralize a portion of the negative charge
which is associated with the molecule of interest (or other
polyanionic materials) present. The amount of cationic lipids used
is typically the amount sufficient to neutralize at least 50% of
the negative charge of the molecule of interest. Preferably, the
negative charge will be at least 70% neutralized, more preferably
at least 90% neutralized. Cationic lipids which are useful in the
present invention include, for example, compounds having any of
formulae CLI-CLXXIX, DODAC, DOTMA, DDAB, DOTAP, DC-Chol, DMOBA,
CLinDMA, and DMRIE. These lipids and related analogs have been
described in U.S. Ser. No. 08/316,399; U.S. Pat. Nos. 5,208,036,
5,264,618, 5,279,833 and 5,283,185, the disclosures of which are
incorporated by reference in their entireties herein. Additionally,
a number of commercial preparations of cationic lipids are
available and can be used in the present invention. These include,
for example, LIPOFECTIN.RTM. (commercially available cationic
liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island,
N.Y., USA); LIPOFECTAMINE.RTM. (commercially available cationic
liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and
TRANSFECTAM.RTM. (commercially available cationic lipids comprising
DOGS in ethanol from Promega Corp., Madison, Wis., USA).
[0520] Contacting the cationic lipids with the molecule-lipid
mixture can be accomplished by any of a number of techniques,
preferably by mixing together a solution of the cationic lipid and
a solution containing the molecule-lipid mixture. Upon mixing the
two solutions (or contacting in any other manner), a portion of the
negative charge associated with the molecule of interest is
neutralized.
[0521] After the cationic lipids have been contacted with the
molecule-lipid mixture, the detergent (or combination of detergent
and organic solvent) is removed, thus forming the formulated
molecular composition. The methods used to remove the detergent
typically involve dialysis. When organic solvents are present,
removal is typically accomplished by evaporation at reduced
pressures or by blowing a stream of inert gas (e.g., nitrogen or
argon) across the mixture.
[0522] The lipid nanoparticle (LNP) composition particles thus
formed are typically sized from about 50 nm to several microns. To
achieve further size reduction or homogeneity of size in the
particles, the lipid nanoparticle (LNP) composition particles can
be sonicated, filtered or subjected to other sizing techniques
which are used in liposomal formulations and are known to those of
skill in the art.
[0523] In other embodiments, the methods further comprise adding
nonlipid polycations which are useful to affect the lipofection of
cells using the present compositions. Examples of suitable nonlipid
polycations include, hexadimethrine bromide (sold under the
brandname 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.
[0524] In another aspect, the present invention provides methods
for the preparation of formulated siNA compositions, comprising:
(a) contacting an amount of cationic lipids with siNA in a
solution; the solution comprising from about 15-35% water and about
65-85% organic solvent and the amount of cationic lipids being
sufficient to produce a .+-. charge ratio of from about 0.85 to
about 2.0, to provide a hydrophobic lipid-siNA complex; (b)
contacting the hydrophobic, lipid-siNA complex in solution with
neutral lipids, to provide a siNA-lipid mixture; and (c) removing
the organic solvents from the lipid-siNA mixture to provide
formulated siNA composition particles.
[0525] The siNA, neutral lipids, cationic lipids and organic
solvents which are useful in this aspect of the invention are the
same as those described for the methods above which used
detergents. In one group of embodiments, the solution of step (a)
is a mono-phase. In another group of embodiments, the solution of
step (a) is two-phase.
[0526] In one embodiment, the cationic lipids used in a formulation
of the invention are selected from a compound having Formula CLI,
CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX, CLX, CLXI,
CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVII, CLXVIII, CLXIX, CLXX,
CLXXI, CLXXII, CLXXIII, CLXXIV, CLXXV, CLXXVI, CLXXVII, CLXXVIII,
CLXXIX, CLXXX, CLXXXI, CLXXXII, CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI,
CLXXXVII, CLXXXVIII, CLXXXIX, CLXXXX, CLXXXXI, CLXXXXII and DODAC,
DDAB, DOTMA, DODAP, DOCDAP, DLINDAP, DOSPA, DMRIE, DOGS, DMOBA,
CLinDMA, and combinations thereof. In one embodiment, the
noncationic lipids are selected from ESM, DOPE, DOPC, DSPC,
polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000 or
PEG-modified diacylglycerols), distearoylphosphatidylcholine
(DSPC), cholesterol, and combinations thereof. In one embodiment,
the organic solvents are selected from methanol, chloroform,
methylene chloride, ethanol, diethyl ether and combinations
thereof.
[0527] In one embodiment, the cationic lipid is a compound having
Formula CLI, CLII, CLIII, CLIV, CLV, CLVI, CLVII, CLVIII, CLIX,
CLX, CLXI, CLXII, CLXIII, CLXIV, CLXV, CLXVI, CLXVII, CLXVIII,
CLXVII, CLXVIII, CLXIX, CLXX, CLXXI, CLXXII, CLXXIII, CLXXIV,
CLXXV, CLLXXVI, CLXXVII, CLXXVIII, CLXXIX, CLXXX, CLXXXI, CLXXXII,
CLXXXIII, CLXXXIV, CLXXXV, CLXXXVI, CLXXXVII, CLXXXVIII, CLXXXIX or
DODAC, DOTAP, DODAP, DOCDAP, DLINDAP, DDAB, DOTMA, DOSPA, DMRIE,
DOGS or combinations thereof; the noncationic lipid is ESM, DOPE,
DAG-PEGs, distearoylphosphatidylcholine (DSPC), cholesterol, or
combinations thereof (e.g. DSPC and DAG-PEGs); and the organic
solvent is methanol, chloroform, methylene chloride, ethanol,
diethyl ether or combinations thereof.
[0528] As above, contacting the siNA with the cationic lipids is
typically accomplished by mixing together a first solution of siNA
and a second solution of the lipids, preferably by mechanical means
such as by using vortex mixers. The resulting mixture contains
complexes as described above. These complexes are then converted to
particles by the addition of neutral lipids and the removal of the
organic solvent. The addition of the neutral lipids is typically
accomplished by simply adding a solution of the neutral lipids to
the mixture containing the complexes. A reverse addition can also
be used. Subsequent removal of organic solvents can be accomplished
by methods known to those of skill in the art and also described
above.
[0529] The amount of neutral lipids which is used in this aspect of
the invention is typically an amount of from about 0.2 to about 15
times the amount (on a mole basis) of cationic lipids which was
used to provide the charge-neutralized lipid-nucleic acid complex.
Preferably, the amount is from about 0.5 to about 9 times the
amount of cationic lipids used.
[0530] In yet another aspect, the present invention provides
formulated siNA compositions which are prepared by the methods
described above. In these embodiments, the formulated siNA
compositions are either net charge neutral or carry an overall
charge which provides the formulated siNA compositions with greater
lipofection activity. In one embodiment, the noncationic lipid is
egg sphingomyelin and the cationic lipid is DODAC. In one
embodiment, the noncationic lipid is a mixture of DSPC and
cholesterol, and the cationic lipid is DOTMA. In another
embodiment, the noncationic lipid can further comprise
cholesterol.
[0531] Non-limiting examples of methods of preparing nucleic acid
formulations are disclosed in U.S. Pat. No. 5,976,567, U.S. Pat.
No. 5,981,501 and PCT Patent Publication No. WO 96/40964, the
teachings of all of which are incorporated in their entireties
herein by reference. Cationic lipids that are useful in the present
invention can be any of a number of lipid species which carry a net
positive charge at a selected pH, such as physiological pH.
Suitable cationic lipids include, but are not limited to, a
compound having any of Formulae CLI-CLXXXXII, DODAC, DOTMA, DDAB,
DOTAP, DODAP, DOCDAP, DLINDAP, DOSPA, DOGS, DC-Chol and DMRIE, as
well as other cationic lipids described herein, or combinations
thereof. A number of these cationic lipids and related analogs,
which are also useful in the present invention, have been described
in U.S. Ser. No. 08/316,399; U.S. Pat. Nos. 5,208,036, 5,264,618,
5,279,833 and 5,283,185, the disclosures of which are incorporated
herein by reference. Additionally, a number of commercial
preparations of cationic lipids are available and can be used in
the present invention. These include, for example, LIPOFECTIN.RTM.
(commercially available cationic liposomes comprising DOTMA and
DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE.RTM.
(commercially available cationic liposomes comprising DOSPA and
DOPE, from GIBCO/BRL); and TRANSFECTAM.RTM. (commercially available
cationic liposomes comprising DOGS from Promega Corp., Madison,
Wis., USA).
[0532] The noncationic lipids used in the present invention can be
any of a variety of neutral uncharged, zwitterionic or anionic
lipids capable of producing a stable complex. They are preferably
neutral, although they can alternatively be positively or
negatively charged. Examples of noncationic lipids useful in the
present invention include phospholipid-related materials, such as
lecithin, phosphatidylethanolamine, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides, dicetylphosphate,
distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylet-hanolamine (POPE) and
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal).
Noncationic lipids or sterols such as cholesterol may be present.
Additional nonphosphorous containing lipids are, 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 and the like,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, and cerebrosides. Other lipids
such as lysophosphatidylcholine and lysophosphatidylethanolamine
may be present. Noncationic lipids also include polyethylene
glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene
glycol conjugated to phospholipids or to ceramides (referred to as
PEG-Cer), as described in co-pending U.S. Ser. No. 08/316,429,
incorporated herein by reference.
[0533] In one embodiment, the noncationic lipids are
diacylphosphatidylcholine (e.g., distearoylphosphatidylcholine,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine or
dilinoleoylphosphatidylcholine), diacylphosphatidylethanolamine
(e.g., dioleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylethanolamine), ceramide or
sphingomyelin. The acyl groups in these lipids are preferably acyl
groups derived from fatty acids having about C10 to about C24
carbon chains. In one embodiment, the acyl groups are lauroyl,
myristoyl, palmitoyl, stearoyl or oleoyl. In additional
embodiments, the noncationic lipid comprises cholesterol,
1,2-sn-dioleoylphosphatidylethanol-amine, or egg sphingomyelin
(ESM).
[0534] In addition to cationic and neutral lipids, the formulated
molecular compositions of the present invention comprise a
polyethyleneglycol (PEG) conjugate. The PEG conjugate can comprise
a diacylglycerol-polyethyleneglycol conjugate, i.e., a DAG-PEG
conjugate. The term "diacylglycerol" refers to a compound having
2-fatty acyl chains, R1 and R2, 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. Diacylglycerols have the
following general formula VIII:
##STR00059##
wherein R1 and R2 are each an alkyl, substituted alkyl, aryl,
substituted aryl, lipid, or a ligand. In one embodiment, R1 and R2
are each independently a C2 to C30 alkyl group.
[0535] In one embodiment, the DAG-PEG conjugate is a
dilaurylglycerol (C12)-PEG conjugate, a dimyristylglycerol
(C14)-PEG conjugate, a dipalmitoylglycerol (C16)-PEG conjugate, a
disterylglycerol (C18)-PEG conjugate, a PEG-dilaurylglycamide
conjugate (C12), a PEG-dimyristylglycamide conjugate (C14), a
PEG-dipalmitoylglycamide conjugate (C16), or a
PEG-disterylglycamide (C18). Those of skill in the art will readily
appreciate that other diacylglycerols can be used in the DAG-PEG
conjugates of the present invention.
[0536] The PEG conjugate can alternatively comprise a conjugate
other than a DAG-PEG conjugate, such as a PEG-cholesterol conjugate
or a PEG-DMB conjugate.
[0537] In addition to the foregoing components, the formulated
molecular compositions or LNPs of the present invention can further
comprise cationic poly(ethylene glycol) (PEG) lipids, or CPLs, that
have been designed for insertion into lipid bilayers to impart a
positive charge (see for example Chen, et al., 2000, Bioconj. Chem.
11, 433-437). Suitable formulations for use in the present
invention, and methods of making and using such formulations are
disclosed, for example in U.S. application Ser. No. 09/553,639,
which was filed Apr. 20, 2000, and PCT Patent Application No. CA
00/00451, which was filed Apr. 20, 2000 and which published as WO
00/62813 on Oct. 26, 2000, the teachings of each of which is
incorporated herein in its entirety by reference.
[0538] The formulated molecular compositions of the present
invention, i.e., those formulated molecular compositions or LNPs
containing DAG-PEG conjugates, can be made using any of a number of
different methods. For example, the lipid-nucleic acid particles
can be produced via hydrophobic siNA-lipid intermediate complexes.
The complexes are preferably charge-neutralized. Manipulation of
these complexes in either detergent-based or organic solvent-based
systems can lead to particle formation in which the nucleic acid is
protected.
[0539] The present invention provides a method of preparing
serum-stable formulated molecular compositions (or lipid
nanoparticles, LNPs), including formulations that undergo
pH-dependent phase transition, in which the biologically active
molecule is encapsulated in a lipid bilayer and is protected from
degradation. Additionally, the formulated particles formed in the
present invention are preferably neutral or negatively-charged at
physiological pH. For in vivo applications, neutral particles are
advantageous, while for in vitro applications the particles are
more preferably negatively charged. This provides the further
advantage of reduced aggregation over the positively-charged
liposome formulations in which a biologically active molecule can
be encapsulated in cationic lipids.
[0540] The formulated particles and LNPs made by the methods of
this invention have a size of about 50 to about 600 nm or more,
with certain of the particles being about 65 to 85 nm. The
particles can be formed by either a detergent dialysis method or by
a modification of a reverse-phase method which utilizes organic
solvents to provide a single phase during mixing of the components.
Without intending to be bound by any particular mechanism of
formation, a biologically active molecule is contacted with a
detergent solution of cationic lipids to form a coated molecular
complex. These coated molecules can aggregate and precipitate.
However, the presence of a detergent reduces this aggregation and
allows the coated molecules to react with excess lipids (typically,
noncationic lipids) to form particles in which the biologically
active molecule is encapsulated in a lipid bilayer. The methods
described below for the formation of formulated molecular
compositions using organic solvents follow a similar scheme.
[0541] In some embodiments, the particles are formed using
detergent dialysis. Thus, the present invention provides a method
for the preparation of serum-stable formulated molecular
compositions (including formulations that undergo pH-dependent
phase transition) comprising: (a) combining a molecule of interest
with cationic lipids in a detergent solution to form a coated
molecule-lipid complex; (b) contacting noncationic lipids with the
coated molecule-lipid complex to form a detergent solution
comprising a molecule-lipid complex and noncationic lipids; and (c)
dialyzing the detergent solution of step (b) to provide a solution
of serum-stable molecule-lipid particles, wherein the molecule of
interest is encapsulated in a lipid bilayer and the particles have
a size of from about 50 to about 600 nm. In one embodiment, the
particles have a size of from about 50 to about 150 nm.
[0542] An initial solution of coated molecule-lipid complexes is
formed, for example, by combining the molecule of interest with the
cationic lipids in a detergent solution.
[0543] In these embodiments, the detergent solution is preferably
an aqueous solution of a neutral detergent having a critical
micelle concentration of 15-300 mM, more preferably 20-50 mM.
Examples of suitable detergents include, for example,
N,N'-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide)
(BIGCHAP); BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol)
ether; Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8;
Mega 9; Zwittergent.RTM. 3-08; Zwittergent.RTM. 3-10; Triton X-405;
hexyl-, heptyl-, octyl- and nonyl-beta-D-glucopyranoside; and
heptylthioglucopyranoside. In one embodiment, the detergent is
octyl .beta.-D-glucopyranoside or Tween-20. The concentration of
detergent in the detergent solution is typically about 100 mM to
about 2 M, preferably from about 200 mM to about 1.5 M.
[0544] The cationic lipids and molecules to be encapsulated will
typically be combined to produce a charge ratio (.+-.) of about 1:1
to about 20:1, preferably in a ratio of about 1:1 to about 12:1,
and more preferably in a ratio of about 2:1 to about 6:1.
Additionally, the overall concentration of the molecules of
interest in solution will typically be from about 25 .mu.g/mL to
about 1 mg/mL, preferably from about 25 .mu.g/mL to about 500
.mu.g/mL, and more preferably from about 100 .mu.g/mL to about 250
.mu.g/mL. The combination of molecules and cationic lipids in
detergent solution is kept, typically at room temperature, for a
period of time which is sufficient for the coated complexes to
form. Alternatively, the molecules and cationic lipids can be
combined in the detergent solution and warmed to temperatures of up
to about 37.degree. C. For molecules which are particularly
sensitive to temperature, the coated complexes can be formed at
lower temperatures, typically down to about 4.degree. C.
[0545] In one embodiment, the molecule to lipid ratios (mass/mass
ratios) in a formed formulated molecular composition will range
from about 0.01 to about 0.08. The ratio of the starting materials
also falls within this range because the purification step
typically removes the unencapsulated molecule as well as the empty
liposomes. In another embodiment, the formulated molecular
composition preparation uses about 400 .mu.g siNA per 10 mg total
lipid or a molecule to lipid 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 siNA.
[0546] The detergent solution of the coated molecule-lipid
complexes is then contacted with neutral lipids to provide a
detergent solution of molecule-lipid complexes and neutral lipids.
The neutral lipids which are useful in this step include, among
others, diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
In preferred embodiments, the neutral lipids are
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide
or sphingomyelin. The acyl groups in these lipids are preferably
acyl groups derived from fatty acids having C10-C24 carbon chains.
More preferably the acyl groups are lauroyl, myristoyl, palmitoyl,
stearoyl or oleoyl. In preferred embodiments, the neutral lipid is
1,2-sn-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl
phosphatidylcholine (POPC), egg phosphatidylcholine (EPC),
distearoylphosphatidylcholine (DSPC), cholesterol, or a mixture
thereof. In the most preferred embodiments, the siNA-lipid
particles are fusogenic particles with enhanced properties in vivo
and the neutral lipid is DSPC or DOPE. As explained above, the
siNA-lipid particles of the present invention can further comprise
PEG conjugates, such as DAG-PEG conjugates, PEG-cholesterol
conjugates, and PEG-DMB conjugates. In addition, the siNA-lipid
particles of the present invention can further comprise
cholesterol.
[0547] The amount of neutral lipid which is used in the present
methods is typically about 0.5 to about 10 mg of total lipids to 50
.mu.g of the molecule of interest. Preferably the amount of total
lipid is from about 1 to about 5 mg per 50 .mu.g of the molecule of
interest.
[0548] Following formation of the detergent solution of
molecule-lipid complexes and neutral lipids, the detergent is
removed, preferably by dialysis. The removal of the detergent
results in the formation of a lipid-bilayer which surrounds the
molecule of interest providing serum-stable molecule-lipid
particles which have a size of from about 50 nm to about 150 or 50
nm to about 600 nm. The particles thus formed do not aggregate and
are optionally sized to achieve a uniform particle size.
[0549] The serum-stable molecule-lipid particles can be sized by
any of the methods available for sizing liposomes as are known in
the art. The sizing can be conducted in order to achieve a desired
size range and relatively narrow distribution of particle
sizes.
[0550] 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, incorporated herein by reference. 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 80 nm, are observed. In both
methods, the particle size distribution can be monitored by
conventional laser-beam particle size discrimination, or QELS.
[0551] 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 can be extruded
through successively smaller-pore membranes, to achieve a gradual
reduction in size.
[0552] A variety of general methods for making formulated siNA
composition-CPLs (CPL-containing formulated siNA compositions) are
discussed herein. Two general techniques include "post-insertion"
technique, that is, insertion of a CPL into for example, a
preformed formulated siNA composition, and the "standard"
technique, wherein the CPL is included in the lipid mixture during
for example, the formulated siNA composition formation steps. The
post-insertion technique results in formulated siNA compositions
having CPLs mainly in the external face of the formulated siNA
composition bilayer membrane, whereas standard techniques provide
formulated siNA compositions having CPLs on both internal and
external faces.
[0553] In particular, "post-insertion" involves forming formulated
siNA compositions (by any method), and incubating the pre-formed
formulated siNA compositions in the presence of CPL under
appropriate conditions (preferably 2-3 hours at 60.degree. C.).
Between 60-80% of the CPL can be inserted into the external leaflet
of the recipient vesicle, giving final concentrations up to about 5
to 10 mol % (relative to total lipid). The method is especially
useful for vesicles made from phospholipids (which can contain
cholesterol) and also for vesicles containing PEG-lipids (such as
PEG-DAGs).
[0554] In an example of a "standard" technique, the CPL-formulated
siNA compositions of the present invention can be formed by
extrusion. In this embodiment, all of the lipids including the CPL,
are co-dissolved in chloroform, which is then removed under
nitrogen followed by high vacuum. The lipid mixture is hydrated in
an appropriate buffer, and extruded through two polycarbonate
filters with a pore size of 100 nm. The resulting formulated siNA
compositions contain CPL on both of the internal and external
faces. In yet another standard technique, the formation of
CPL-formulated siNA compositions can be accomplished using a
detergent dialysis or ethanol dialysis method, for example, as
discussed in U.S. Pat. Nos. 5,976,567 and 5,981,501, both of which
are incorporated by reference in their entireties herein.
[0555] The formulated siNA compositions of the present invention
can be administered either alone or in mixture with a
physiologically-acceptable carrier (such as physiological saline or
phosphate buffer) selected in accordance with the route of
administration and standard pharmaceutical practice. Generally,
normal saline 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.
[0556] The pharmaceutical carrier is generally added following
formulated siNA composition formation. Thus, after the formulated
siNA composition is formed, the formulated siNA composition can be
diluted into pharmaceutically acceptable carriers such as normal
saline.
[0557] The concentration of formulated siNA compositions in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.05%, usually at or at least about 2-5% to as much as 10 to
30% 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 can 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, formulated siNA compositions composed
of irritating lipids can be diluted to low concentrations to lessen
inflammation at the site of administration.
[0558] As described above, the formulated siNA compositions of the
present invention comprise DAG-PEG conjugates. It is often
desirable to include other components that act in a manner similar
to the DAG-PEG conjugates and that serve to prevent particle
aggregation and to provide a means for increasing circulation
lifetime and increasing the delivery of the formulated siNA
compositions to the target tissues. Such components include, but
are not limited to, PEG-lipid conjugates, such as PEG-ceramides or
PEG-phospholipids (such as PEG-PE), ganglioside GM1-modified lipids
or ATTA-lipids to the particles. Typically, the concentration of
the component in the particle will be about 1-20% and, more
preferably from about 3-10%.
[0559] The pharmaceutical compositions of the present invention can
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 can 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
[0560] In another example of their use, formulated molecular
compositions can be incorporated into a broad range of topical
dosage forms including, but not limited to, gels, oils, emulsions
and the like. For instance, the suspension containing the
formulated molecular compositions can be formulated and
administered as topical creams, pastes, ointments, gels, lotions
and the like.
[0561] Once formed, the formulated molecular compositions of the
present invention are useful for the introduction of biologically
active molecules into cells. Accordingly, the present invention
also provides methods for introducing a biologically active
molecule into a cell. The methods are carried out in vitro or in
vivo by first forming the formulated molecular compositions as
described above and then contacting the formulated molecular
compositions with the cells for a period of time sufficient for
transfection to occur.
[0562] The formulated molecular compositions of the present
invention can be adsorbed to almost any cell type with which they
are mixed or contacted. Once adsorbed, the formulations 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
biologically acitive molecule portion of the formulation 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, i.e., biologically
active molecules, combine with the intracellular fluid, for
example, the cytoplasm. The serum stable formulated molecular
compositions that undergo pH-dependent phase transition demonstrate
an increase in cell fusion at early endosomal pH (i.e., about pH
5.5-6.5), resulting in efficient delivery of the contents of the
particle, i.e., biologically active molecules, to the cell.
[0563] Using the Endosomal Release Parameter (ERP) assay of the
present invention, the transfection efficiency of the formulated
molecular composition or other lipid-based carrier system can be
optimized. More particularly, the purpose of the ERP assay is to
distinguish the effect of various cationic lipids and helper lipid
components of formulated molecular compositions 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 formulated molecular
composition or other lipid-based carrier system effects
transfection efficacy, thereby optimizing the formulated molecular
compositions or other lipid-based carrier systems. As explained
herein, the Endosomal Release Parameter or, alternatively, ERP is
defined as: Reporter Gene Expression/Cell divided by formulated
molecular composition Uptake/Cell.
[0564] It will be readily apparent to those of skill in the art
that any reporter gene (e.g., luciferase, beta-galactosidase, green
fluorescent protein, etc.) can be used in the assay. In addition,
the lipid component (or, alternatively, any component of the
formulated molecular composition) can be labeled with any
detectable label provided the does inhibit or interfere with uptake
into the cell. Using the ERP assay of the present invention, one of
skill in the art can assess the impact of the various lipid
components (e.g., cationic lipid, neutral lipid, PEG-lipid
derivative, PEG-DAG conjugate, ATTA-lipid derivative, calcium,
CPLs, cholesterol, etc.) on cell uptake and transfection
efficiencies, thereby optimizing the formulated siNA composition.
By comparing the ERPs for each of the various formulated molecular
compositions, one can readily determine the optimized system, e.g.,
the formulated molecular composition that has the greatest uptake
in the cell coupled with the greatest transfection efficiency.
[0565] Suitable labels for carrying out the ERP assay of the
present invention include, but are not limited to, spectral labels,
such as fluorescent dyes (e.g., fluorescein and derivatives, such
as fluorescein isothiocyanate (FITC) and Oregon Green9; rhodamine
and derivatives, such Texas red, tetrarhodimine isothiocynate
(TRITC), etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes,
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
coupled directly or indirectly to a component of the formulated
molecular composition using methods well known in the art. As
indicated above, a wide variety of labels can be used, with the
choice of label depending on sensitivity required, ease of
conjugation with the formulated siNA composition, stability
requirements, and available instrumentation and disposal
provisions.
[0566] In addition, the transfection efficiency of the formulated
molecular composition or other lipid-based carrier system can be
determined by measuring the stability of the composition in serm
and/or measuring the pH dependent phase transition of the
formulated molecular composition, wherein a determination that the
formulated molecular composition is stable in serum and a
determination that the formulated molecular composition undergoes a
phase transition at about pH 5.5-6.5 indicates that the formulated
molecular composition will have increased transfection efficiency.
The serum stability of the formulated molecular composition can be
measured using, for example, an assay that measures the relative
turbidity of the composition in serum and determining that the
turbity of the composition in serum remains constant over time. The
pH dependent phase transition of the formulated molecular
composition can be measured using an assay that measures the
relative turbidity of the composition at different pH over time and
determining that the turbidity changes when the pH differs from
physiologic pH.
Optimizing Activity of the Nucleic Acid Molecule of the
Invention.
[0567] Chemically synthesizing nucleic acid molecules (e.g., siNA,
miRNA, RNAi inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A,
triplex forming oligonucleotide, or other nucleic acid molecule)
with modifications (base, sugar and/or phosphate) can prevent their
degradation by serum ribonucleases, which can increase their
potency (see e.g., Eckstein et al., International Publication No.
WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al.,
1991, Science 253, 314; Usman and Cedergren, 1992, Trends in
Biochem. Sci. 17, 334; Usman et al., International Publication No.
WO 93/15187; and Rossi et al., International Publication No. WO
91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat.
No. 6,300,074; and Burgin et al., supra; all of which are
incorporated by reference herein). All of the above references
describe various chemical modifications that can be made to the
base, phosphate and/or sugar moieties of the nucleic acid molecules
described herein. Modifications that enhance their efficacy in
cells, and removal of bases from nucleic acid molecules to shorten
oligonucleotide synthesis times and reduce chemical requirements
are desired.
[0568] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide
base modifications (for a review see Usman and Cedergren, 1992,
TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163;
Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification
of nucleic acid molecules have been extensively described in the
art (see Eckstein et al., International Publication PCT No. WO
92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.
Science, 1991, 253, 314-317; Usman and Cedergren, Trends in
Biochem. Sci., 1992, 17, 334-339; Usman et al. International
Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711
and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman
et al., International PCT publication No. WO 97/26270; Beigelman et
al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No.
5,627,053; Woolf et al., International PCT Publication No. WO
98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed
on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39,
1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences),
48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,
99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010;
all of the references are hereby incorporated in their totality by
reference herein). Such publications describe general methods and
strategies to determine the location of incorporation of sugar,
base and/or phosphate modifications and the like into nucleic acid
molecules without modulating catalysis, and are incorporated by
reference herein. In view of such teachings, similar modifications
can be used as described herein to modify the siNA nucleic acid
molecules of the instant invention so long as the ability of siNA
to promote RNAi cells is not significantly inhibited.
[0569] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorodithioate,
and/or 5'-methylphosphonate linkages improves stability, excessive
modifications can cause some toxicity or decreased activity.
Therefore, when designing nucleic acid molecules, the amount of
these internucleotide linkages should be minimized. The reduction
in the concentration of these linkages should lower toxicity,
resulting in increased efficacy and higher specificity of these
molecules.
[0570] Polynucleotides (e.g., siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule) having chemical
modifications that maintain or enhance activity are provided. Such
a nucleic acid is also generally more resistant to nucleases than
an unmodified nucleic acid. Accordingly, the in vitro and/or in
vivo activity should not be significantly lowered. In cases in
which modulation is the goal, therapeutic nucleic acid molecules
delivered exogenously should optimally be stable within cells until
translation of the target RNA has been modulated long enough to
reduce the levels of the undesirable protein. This period of time
varies between hours to days depending upon the disease state.
Improvements in the chemical synthesis of RNA and DNA (Wincott et
al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,
Methods in Enzymology 211, 3-19 (incorporated by reference herein))
have expanded the ability to modify nucleic acid molecules by
introducing nucleotide modifications to enhance their nuclease
stability, as described above.
[0571] In one embodiment, nucleic acid molecules of the invention
include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more) G-clamp nucleotides. A G-clamp nucleotide is a modified
cytosine analog wherein the modifications confer the ability to
hydrogen bond both Watson-Crick and Hoogsteen faces of a
complementary guanine within a duplex, see for example Lin and
Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. A single
G-clamp analog substitution within an oligonucleotide can result in
substantially enhanced helical thermal stability and mismatch
discrimination when hybridized to complementary oligonucleotides.
The inclusion of such nucleotides in nucleic acid molecules of the
invention results in both enhanced affinity and specificity to
nucleic acid targets, complementary sequences, or template strands.
In another embodiment, nucleic acid molecules of the invention
include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more) LNA "locked nucleic acid" nucleotides such as a 2',4'-C
methylene bicyclo nucleotide (see for example Wengel et al.,
International PCT Publication No. WO 00/66604 and WO 99/14226).
[0572] In another embodiment, the invention features conjugates
and/or complexes of siNA molecules of the invention. Such
conjugates and/or complexes can be used to facilitate delivery of
siNA molecules into a biological system, such as a cell. The
conjugates and complexes provided by the instant invention can
impart therapeutic activity by transferring therapeutic compounds
across cellular membranes, altering the pharmacokinetics, and/or
modulating the localization of nucleic acid molecules of the
invention. The present invention encompasses the design and
synthesis of novel conjugates and complexes for the delivery of
molecules, including, but not limited to, small molecules, lipids,
cholesterol, phospholipids, nucleosides, nucleotides, nucleic
acids, antibodies, toxins, negatively charged polymers and other
polymers, for example proteins, peptides, hormones, carbohydrates,
polyethylene glycols, or polyamines, across cellular membranes. In
general, the transporters described are designed to be used either
individually or as part of a multi-component system, with or
without degradable linkers. These compounds are expected to improve
delivery and/or localization of nucleic acid molecules of the
invention into a number of cell types originating from different
tissues, in the presence or absence of serum (see Sullenger and
Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules
described herein can be attached to biologically active molecules
via linkers that are biodegradable, such as biodegradable nucleic
acid linker molecules.
[0573] The term "biodegradable linker" as used herein, refers to a
nucleic acid or non-nucleic acid linker molecule that is designed
as a biodegradable linker to connect one molecule to another
molecule, for example, a biologically active molecule to a siNA
molecule of the invention or the sense and antisense strands of a
siNA molecule of the invention. The biodegradable linker is
designed such that its stability can be modulated for a particular
purpose, such as delivery to a particular tissue or cell type. The
stability of a nucleic acid-based biodegradable linker molecule can
be modulated by using various chemistries, for example combinations
of ribonucleotides, deoxyribonucleotides, and chemically-modified
nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino,
2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified
nucleotides. The biodegradable nucleic acid linker molecule can be
a dimer, trimer, tetramer or longer nucleic acid molecule, for
example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or
can comprise a single nucleotide with a phosphorus-based linkage,
for example, a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise
nucleic acid backbone, nucleic acid sugar, or nucleic acid base
modifications.
[0574] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example, enzymatic
degradation or chemical degradation.
[0575] The term "phospholipid" as used herein, refers to a
hydrophobic molecule comprising at least one phosphorus group. For
example, a phospholipid can comprise a phosphorus-containing group
and saturated or unsaturated alkyl group, optionally substituted
with OH, COOH, oxo, amine, or substituted or unsubstituted aryl
groups.
[0576] Therapeutic nucleic acid molecules (e.g., siNA, miRNA, RNAi
inhibitor, antisense, aptamer, decoy, ribozyme, 2-5A, triplex
forming oligonucleotide, or other nucleic acid molecule) delivered
exogenously optimally are stable within cells until reverse
transcription of the RNA has been modulated long enough to reduce
the levels of the RNA transcript. The nucleic acid molecules are
resistant to nucleases in order to function as effective
intracellular therapeutic agents. Improvements in the chemical
synthesis of nucleic acid molecules described in the instant
invention and in the art have expanded the ability to modify
nucleic acid molecules by introducing nucleotide modifications to
enhance their nuclease stability as described above.
[0577] In yet another embodiment, siNA molecules having chemical
modifications that maintain or enhance enzymatic activity of
proteins involved in RNAi are provided. Such nucleic acids are also
generally more resistant to nucleases than unmodified nucleic
acids. Thus, in vitro and/or in vivo the activity should not be
significantly lowered.
[0578] Use of the nucleic acid-based molecules of the invention
will lead to better treatments by affording the possibility of
combination therapies (e.g., multiple siNA molecules targeted to
different genes; nucleic acid molecules coupled with known small
molecule modulators; or intermittent treatment with combinations of
molecules, including different motifs and/or other chemical or
biological molecules).
[0579] In another aspect a polynucleotide molecule of the invention
(e.g., siNA, miRNA, RNAi inhibitor, antisense, aptamer, decoy,
ribozyme, 2-5A, triplex forming oligonucleotide, or other nucleic
acid molecule) comprises one or more 5' and/or a 3'-cap structure,
for example, on only the sense siNA strand, the antisense siNA
strand, or both siNA strands.
[0580] By "cap structure" is meant chemical modifications, which
have been incorporated at either terminus of the oligonucleotide
(see, for example, Adamic et al., U.S. Pat. No. 5,998,203,
incorporated by reference herein). These terminal modifications
protect the nucleic acid molecule from exonuclease degradation, and
may help in delivery and/or localization within a cell. The cap may
be present at the 5'-terminus (5'-cap) or at the 3'-terminal
(3'-cap) or may be present on both termini. In non-limiting
examples, the 5'-cap includes, but is not limited to, glyceryl,
inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety
[0581] Non-limiting examples of the 3'-cap include, but are not
limited to, glyceryl, inverted deoxy abasic residue (moiety),
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide;
4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl
phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0582] By the term "non-nucleotide" is meant any group or compound
which can be incorporated into a nucleic acid chain in the place of
one or more nucleotide units, including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit
their enzymatic 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 1'-position.
[0583] An "alkyl" group refers to a saturated aliphatic
hydrocarbon, including straight-chain, branched-chain, and cyclic
alkyl groups. Preferably, and unless expressly stated to to the
contrary, the alkyl group has 1 to 12 carbons. More preferably, it
is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4
carbons. The alkyl group can be substituted or unsubstituted. When
substituted the substituted group(s) is preferably, hydroxyl,
cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or N(CH.sub.3).sub.2,
amino, or SH. The term also includes alkenyl groups that are
unsaturated hydrocarbon groups containing at least one
carbon-carbon double bond, including straight-chain,
branched-chain, and cyclic groups. Preferably, the alkenyl group
has 1 to 12 carbons. More preferably, it is a lower alkenyl of from
1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group
may be substituted or unsubstituted. When substituted the
substituted group(s) is preferably, hydroxyl, cyano, alkoxy,
.dbd.O, .dbd.S, NO.sub.2, halogen, N(CH.sub.3).sub.2, amino, or SH.
The term "alkyl" also includes alkynyl groups that have an
unsaturated hydrocarbon group containing at least one carbon-carbon
triple bond, including straight-chain, branched-chain, and cyclic
groups. Preferably, the alkynyl group has 1 to 12 carbons. More
preferably, it is a lower alkynyl of from 1 to 7 carbons, more
preferably 1 to 4 carbons. The alkynyl group may be substituted or
unsubstituted. When substituted the substituted group(s) is
preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or
N(CH.sub.3).sub.2, amino or SH.
[0584] Such alkyl groups can also include aryl, alkylaryl,
carbocyclic aryl, heterocyclic aryl, amide and ester groups. An
"aryl" group refers to an aromatic group that has at least one ring
having a conjugated pi electron system and includes carbocyclic
aryl, heterocyclic aryl and biaryl groups, all of which may be
optionally substituted. The preferred substituent(s) of aryl groups
are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl,
alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to
an alkyl group (as described above) covalently joined to an aryl
group (as described above). Carbocyclic aryl groups are groups
wherein the ring atoms on the aromatic ring are all carbon atoms.
The carbon atoms are optionally substituted. Heterocyclic aryl
groups are groups having from 1 to 3 heteroatoms as ring atoms in
the aromatic ring and the remainder of the ring atoms are carbon
atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,
and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl
pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all
optionally substituted. An "amide" refers to an --C(O)--NH--R,
where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester"
refers to an --C(O)--OR', where R is either alkyl, aryl, alkylaryl
or hydrogen.
[0585] By "nucleotide" as used herein is as recognized in the art
to include natural bases (standard), and modified bases well known
in the art. Such bases are generally located at the 1' position of
a nucleotide sugar moiety. Nucleotides generally comprise a base,
sugar and a phosphate group. The nucleotides can be unmodified or
modified at the sugar, phosphate and/or base moiety, (also referred
to interchangeably as nucleotide analogs, modified nucleotides,
non-natural nucleotides, non-standard nucleotides and other; see,
for example, Usman and McSwiggen, supra; Eckstein et al.,
International PCT Publication No. WO 92/07065; Usman et al.,
International PCT Publication No. WO 93/15187; Uhlman & Peyman,
supra, all are hereby incorporated by reference herein). There are
several examples of modified nucleic acid bases known in the art as
summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
Some of the non-limiting examples of base modifications that can be
introduced into nucleic acid molecules include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,
naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),
5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
6-methyluridine), propyne, and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified
bases" in this aspect is meant nucleotide bases other than adenine,
guanine, cytosine and uracil at 1' position or their
equivalents.
[0586] In one embodiment, the invention features modified
polynucleotide molecules (e.g., siNA, miRNA, RNAi inhibitor,
antisense, aptamer, decoy, ribozyme, 2-5A, triplex forming
oligonucleotide, or other nucleic acid molecule), with phosphate
backbone modifications comprising one or more phosphorothioate,
phosphorodithioate, methylphosphonate, phosphotriester, morpholino,
amidate carbamate, carboxymethyl, acetamidate, polyamide,
sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal,
and/or alkylsilyl, substitutions. For a review of oligonucleotide
backbone modifications, see Hunziker and Leumann, 1995, Nucleic
Acid Analogues: Synthesis and Properties, in Modern Synthetic
Methods, VCH, 331-417, and Mesmaeker et al., 1994, Novel Backbone
Replacements for Oligonucleotides, in Carbohydrate Modifications in
Antisense Research, ACS, 24-39.
[0587] By "abasic" is meant sugar moieties lacking a base or having
other chemical groups in place of a base at the 1' position, see
for example Adamic et al., U.S. Pat. No. 5,998,203.
[0588] By "unmodified nucleoside" is meant one of the bases
adenine, cytosine, guanine, thymine, or uracil joined to the 1'
carbon of .beta.-D-ribo-furanose.
[0589] By "modified nucleoside" is meant any nucleotide base which
contains a modification in the chemical structure of an unmodified
nucleotide base, sugar and/or phosphate. Non-limiting examples of
modified nucleotides are shown by Formulae I-VII and/or other
modifications described herein.
[0590] In connection with 2'-modified nucleotides as described for
the present invention, by "amino" is meant 2'-NH.sub.2 or
2'-O--NH.sub.2, which can be modified or unmodified. Such modified
groups are described, for example, in Eckstein et al., U.S. Pat.
No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878,
which are both incorporated by reference in their entireties.
[0591] Various modifications to nucleic acid siNA structure can be
made to enhance the utility of these molecules. Such modifications
will enhance shelf-life, half-life in vitro, stability, and ease of
introduction of such oligonucleotides to the target site, e.g., to
enhance penetration of cellular membranes, and confer the ability
to recognize and bind to targeted cells.
[0592] By "cholesterol derivative" is meant, any compound
consisting essentially of a cholesterol structure, including
additions, substitutions and/or deletions thereof. The term
cholesterol derivative herein also includes steroid hormones and
bile acids as are generally recognized in the art.
Administration of Formulated siNA Compositions
[0593] A formulated molecular composition of the invention can be
adapted for use to prevent, inhibit, or reduce any trait, disease
or condition that is related to or will respond to the levels of
target gene expression in a cell or tissue, alone or in combination
with other therapies.
[0594] In one embodiment, formulated molecular compositions can be
administered to cells by a variety of methods known to those of
skill in the art, including, but not restricted to, by injection,
by iontophoresis or by incorporation into other vehicles, such as
biodegradable polymers, hydrogels, cyclodextrins (see for example
Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et
al., International PCT publication Nos. WO 03/47518 and WO
03/46185). In one embodiment, a formulated molecular compositions
of the invention are complexed with membrane disruptive agents such
as those described in U.S. Patent Application Publication No.
20010007666, incorporated by reference herein in its entirety
including the drawings. In another embodiment, the membrane
disruptive agent or agents and the biologically active molecule are
also complexed with a cationic lipid or helper lipid molecule, such
as those lipids described in U.S. Pat. No. 6,235,310, incorporated
by reference herein in its entirety including the drawings.
[0595] In one embodiment, delivery systems of the invention
include, for example, aqueous and nonaqueous gels, creams, multiple
emulsions, microemulsions, ointments, aqueous and nonaqueous
solutions, lotions, aerosols, hydrocarbon bases and powders, and
can contain excipients such as solubilizers, permeation enhancers
(e.g., fatty acids, fatty acid esters, fatty alcohols and amino
acids), and hydrophilic polymers (e.g., polycarbophil and
polyvinylpyrolidone). In one embodiment, the pharmaceutically
acceptable carrier is a transdermal enhancer.
[0596] In one embodiment, delivery systems of the invention include
patches, tablets, suppositories, pessaries, gels and creams, and
can contain excipients such as solubilizers and enhancers (e.g.,
propylene glycol, bile salts and amino acids), and other vehicles
(e.g., polyethylene glycol, fatty acid esters and derivatives, and
hydrophilic polymers such as hydroxypropylmethylcellulose and
hyaluronic acid).
[0597] In one embodiment, the invention features a pharmaceutical
composition comprising one or more formulated siNA compositions of
the invention in an acceptable carrier, such as a stabilizer,
buffer, and the like. The formulated molecular compositions of the
invention can be administered and introduced to a subject by any
standard means, with or without stabilizers, buffers, and the like,
to form a pharmaceutical composition. The compositions of the
present invention can also be formulated and used as creams, gels,
sprays, oils and other suitable compositions for topical, dermal,
or transdermal administration as is known in the art.
[0598] In one embodiment, the invention also includes
pharmaceutically acceptable formulations of the compounds
described. These formulations include salts of the above compounds,
e.g., acid addition salts, for example, salts of hydrochloric,
hydrobromic, acetic acid, and benzene sulfonic acid.
[0599] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic or local administration, into a cell or subject,
including for example a human. Suitable forms, in part, depend upon
the use or the route of entry, for example oral, transdermal, or by
injection. Such forms should not prevent the composition or
formulation from reaching a target cell (i.e., a cell to which the
siNA is desirable for delivery). For example, pharmacological
compositions injected into the blood stream should be soluble.
Other factors are known in the art, and include considerations such
as toxicity and forms that prevent the composition or formulation
from exerting its effect.
[0600] In one embodiment, formulated molecular compositions of the
invention are administered to a subject by systemic administration
in a pharmaceutically acceptable composition or formulation. By
"systemic administration" is meant in vivo systemic absorption or
accumulation of drugs in the blood stream followed by distribution
throughout the entire body. Administration routes that lead to
systemic absorption include, without limitation: intravenous,
subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and
intramuscular. Each of these administration routes exposes the siNA
molecules of the invention to an accessible diseased tissue. The
rate of entry of a drug into the circulation has been shown to be a
function of molecular weight or size.
[0601] By "pharmaceutically acceptable formulation" or
"pharmaceutically acceptable composition" is meant, a composition
or formulation that allows for the effective distribution of the
formulated molecular A compositions of the instant invention in the
physical location most suitable for their desired activity.
Non-limiting examples of agents suitable for formulation with the
formulated molecular compositions of the instant invention include:
P-glycoprotein inhibitors (such as Pluronic P85); biodegradable
polymers, such as poly (DL-lactide-coglycolide) microspheres for
sustained release delivery (Emerich, D F et al, 1999, Cell
Transplant, 8, 47-58); and loaded nanoparticles, such as those made
of polybutylcyanoacrylate. Other non-limiting examples of delivery
strategies for the nucleic acid molecules of the instant invention
include material described in Boado et al., 1998, J. Pharm. Sci.,
87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284;
Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv.
Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998,
Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999, PNAS
USA., 96, 7053-7058.
[0602] The present invention also includes compositions prepared
for storage or administration that include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated
by reference herein. For example, preservatives, stabilizers, dyes
and flavoring agents can be provided. These include sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In
addition, antioxidants and suspending agents can be used.
[0603] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence, or treat (alleviate a symptom to
some extent, preferably all of the symptoms) of a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors that those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon
potency of the formulated siNA composition.
[0604] The formulated molecular compositions of the invention can
be administered orally, topically, parenterally, by inhalation or
spray, or rectally in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and/or vehicles. The term parenteral as used herein
includes percutaneous, subcutaneous, intravascular (e.g.,
intravenous), intramuscular, or intrathecal injection or infusion
techniques and the like. In addition, there is provided a
pharmaceutical formulation comprising a formulated molecular
composition of the invention and a pharmaceutically acceptable
carrier. One or more formulated molecular compositions of the
invention can be present in association with one or more non-toxic
pharmaceutically acceptable carriers and/or diluents and/or
adjuvants, and if desired other active ingredients. The
pharmaceutical compositions containing formulated molecular
compositions of the invention can be in a form suitable for oral
use, for example, as tablets, troches, lozenges, aqueous or oily
suspensions, dispersible powders or granules, emulsion, hard or
soft capsules, or syrups or elixirs.
[0605] Compositions intended for oral use can be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions can contain one
or more such sweetening agents, flavoring agents, coloring agents
or preservative agents in order to provide pharmaceutically elegant
and palatable preparations. Tablets contain the active ingredient
in admixture with non-toxic pharmaceutically acceptable excipients
that are suitable for the manufacture of tablets. These excipients
can be, for example, inert diluents; such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia; and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets can be uncoated or they can be
coated by known techniques. In some cases such coatings can be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate can be
employed.
[0606] Formulations for oral use can also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0607] Aqueous suspensions contain the active materials in a
mixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0608] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0609] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0610] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0611] Syrups and elixirs can be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations can also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions can be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension can be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that can be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil can be
employed including synthetic mono-or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0612] The formulated molecular compositions of the invention can
also be administered in the form of suppositories, e.g., for rectal
administration of the drug. These compositions can be prepared by
mixing the drug with a suitable non-irritating excipient that is
solid at ordinary temperatures but liquid at the rectal temperature
and will therefore melt in the rectum to release the drug. Such
materials include cocoa butter and polyethylene glycols.
[0613] Formulated molecular compositions of the invention can be
administered parenterally in a sterile medium. The drug, depending
on the vehicle and concentration used, can either be suspended or
dissolved in the vehicle. Advantageously, adjuvants such as local
anesthetics, preservatives and buffering agents can be dissolved in
the vehicle.
[0614] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
subject per day). The amount of active ingredient that can be
combined with the carrier materials to produce a single dosage form
varies depending upon the host treated and the particular mode of
administration. Dosage unit forms generally contain between from
about 1 mg to about 500 mg of an active ingredient.
[0615] It is understood that the specific dose level for any
particular subject depends upon a variety of factors including the
activity of the specific compound employed, the age, body weight,
general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0616] For administration to non-human animals, the composition can
also be added to the animal feed or drinking water. It can be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It can
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0617] The formulated molecular compositions of the present
invention can also be administered to a subject in combination with
other therapeutic compounds to increase the overall therapeutic
effect. The use of multiple compounds to treat an indication can
increase the beneficial effects while reducing the presence of side
effects.
Examples
[0618] The following are non-limiting examples showing the
selection, isolation, synthesis and activity of nucleic acids of
the instant invention.
Example 1
Identification of Potential siNA Target Sites in any RNA
Sequence
[0619] The sequence of an RNA target of interest, such as a viral
or human mRNA transcript, is screened for target sites, for example
by using a computer folding algorithm. In a non-limiting example,
the sequence of a gene or RNA gene transcript derived from a
database, such as Genbank, is used to generate siNA targets having
complementarity to the target. Such sequences can be obtained from
a database, or can be determined experimentally as known in the
art. Target sites that are known, for example, those target sites
determined to be effective target sites based on studies with other
nucleic acid molecules, for example ribozymes or antisense, or
those targets known to be associated with a disease or condition
such as those sites containing mutations or deletions, can be used
to design siNA molecules targeting those sites. Various parameters
can be used to determine which sites are the most suitable target
sites within the target RNA sequence. These parameters include but
are not limited to secondary or tertiary RNA structure, the
nucleotide base composition of the target sequence, the degree of
homology between various regions of the target sequence, or the
relative position of the target sequence within the RNA transcript.
Based on these determinations, any number of target sites within
the RNA transcript can be chosen to screen siNA molecules for
efficacy, for example by using in vitro RNA cleavage assays, cell
culture, or animal models. In a non-limiting example, anywhere from
1 to 1000 target sites are chosen within the transcript based on
the size of the siNA construct to be used. High throughput
screening assays can be developed for screening siNA molecules
using methods known in the art, such as with multi-well or
multi-plate assays to determine efficient reduction in target gene
expression. These methods can also be used to determine target
sites for, example, antisense, ribozyme, 2-5-A, triplex, and decoy
nucleic acid molecules of the invention.
Example 2
Selection of siNA Molecule Target Sites in a RNA
[0620] The following non-limiting steps can be used to carry out
the selection of siNAs targeting a given gene sequence or
transcript.
[0621] 1. The target sequence is parsed in silico into a list of
all fragments or subsequences of a particular length, for example
23 nucleotide fragments, contained within the target sequence. This
step is typically carried out using a custom Perl script, but
commercial sequence analysis programs such as Oligo, MacVector, or
the GCG Wisconsin Package can be employed as well. [0622] 2. In
some instances the siNAs correspond to more than one target
sequence; such would be the case for example in targeting different
transcripts of the same gene, targeting different transcripts of
more than one gene, or for targeting both the human gene and an
animal homolog. In this case, a subsequence list of a particular
length is generated for each of the targets, and then the lists are
compared to find matching sequences in each list. The subsequences
are then ranked according to the number of target sequences that
contain the given subsequence; the goal is to find subsequences
that are present in most or all of the target sequences.
Alternately, the ranking can identify subsequences that are unique
to a target sequence, such as a mutant target sequence. Such an
approach would enable the use of siNA to target specifically the
mutant sequence and not effect the expression of the normal
sequence. [0623] 3. In some instances the siNA subsequences are
absent in one or more sequences while present in the desired target
sequence; such would be the case if the siNA targets a gene with a
paralogous family member that is to remain untargeted. As in case 2
above, a subsequence list of a particular length is generated for
each of the targets, and then the lists are compared to find
sequences that are present in the target gene but are absent in the
untargeted paralog. [0624] 4. The ranked siNA subsequences can be
further analyzed and ranked according to GC content. A preference
can be given to sites containing 30-70% GC, with a further
preference to sites containing 40-60% GC. [0625] 5. The ranked siNA
subsequences can be further analyzed and ranked according to
self-folding and internal hairpins. Weaker internal folds are
preferred; strong hairpin structures are to be avoided. [0626] 6.
The ranked siNA subsequences can be further analyzed and ranked
according to whether they have runs of GGG or CCC in the sequence.
GGG (or even more Gs) in either strand can make oligonucleotide
synthesis problematic and can potentially interfere with RNAi
activity, so it is avoided whenever better sequences are available.
CCC is searched in the target strand because that will place GGG in
the antisense strand. [0627] 7. The ranked siNA subsequences can be
further analyzed and ranked according to whether they have the
dinucleotide UU (uridine dinucleotide) on the 3'-end of the
sequence, and/or AA on the 5'-end of the sequence (to yield 3' UU
on the antisense sequence). These sequences allow one to design
siNA molecules with terminal TT thymidine dinucleotides. [0628] 8.
Four or five target sites are chosen from the ranked list of
subsequences as described above. For example, in subsequences
having 23 nucleotides, the right 21 nucleotides of each chosen
23-mer subsequence are then designed and synthesized for the upper
(sense) strand of the siNA duplex, while the reverse complement of
the left 21 nucleotides of each chosen 23-mer subsequence are then
designed and synthesized for the lower (antisense) strand of the
siNA duplex. If terminal TT residues are desired for the sequence
(as described in paragraph 7), then the two 3' terminal nucleotides
of both the sense and antisense strands are replaced by TT prior to
synthesizing the oligos. [0629] 9. The siNA molecules are screened
in an in vitro, cell culture or animal model system to identify the
most active siNA molecule or the most preferred target site within
the target RNA sequence. [0630] 10. Other design considerations can
be used when selecting target nucleic acid sequences, see, for
example, Reynolds et al., 2004, Nature Biotechnology Advanced
Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et
al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.
Example 3
siNA Design
[0631] siNA target sites were chosen by analyzing sequences of the
target RNA target and optionally prioritizing the target sites on
the basis of folding (structure of any given sequence analyzed to
determine siNA accessibility to the target), by using a library of
siNA molecules, or alternately by using an in vitro siNA system as
described herein. siNA molecules are designed that could bind each
target and are optionally individually analyzed by computer folding
to assess whether the siNA molecule can interact with the target
sequence. Varying the length of the siNA molecules can be chosen to
optimize activity. Generally, a sufficient number of complementary
nucleotide bases are chosen to bind to, or otherwise interact with,
the target RNA, but the degree of complementarity can be modulated
to accommodate siNA duplexes or varying length or base composition.
By using such methodologies, siNA molecules can be designed to
target sites within any known RNA sequence, for example those RNA
sequences corresponding to the any gene transcript.
[0632] Chemically modified siNA constructs are designed to provide
nuclease stability for systemic administration in vivo and/or
improved pharmacokinetic, localization, and delivery properties
while preserving the ability to mediate RNAi activity. Chemical
modifications as described herein are introduced synthetically
using synthetic methods described herein and those generally known
in the art. The synthetic siNA constructs are then assayed for
nuclease stability in serum and/or cellular/tissue extracts (e.g.
liver extracts). The synthetic siNA constructs are also tested in
parallel for RNAi activity using an appropriate assay, such as a
luciferase reporter assay as described herein or another suitable
assay that can quantity RNAi activity. Synthetic siNA constructs
that possess both nuclease stability and RNAi activity can be
further modified and re-evaluated in stability and activity assays.
The chemical modifications of the stabilized active siNA constructs
can then be applied to any siNA sequence targeting any chosen RNA
and used, for example, in target screening assays to pick lead siNA
compounds for therapeutic development.
Example 4
Chemical Synthesis and Purification of siNA
[0633] siNA molecules can be designed to interact with various
sites in the RNA message, for example, target sequences within the
RNA sequences described herein. The sequence of one strand of the
siNA molecule(s) is complementary to the target site sequences
described above. The siNA molecules can be chemically synthesized
using methods described herein. Inactive siNA molecules that are
used as control sequences can be synthesized by scrambling the
sequence of the siNA molecules such that it is not complementary to
the target sequence. Generally, siNA constructs can by synthesized
using solid phase oligonucleotide synthesis methods as described
herein (see for example Usman et al., U.S. Pat. Nos. 5,804,683;
5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323; 6,437,117;
6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086; 6,008,400;
6,111,086 all incorporated by reference in their entireties
herein).
[0634] In a non-limiting example, RNA oligonucleotides are
synthesized in a stepwise fashion using the phosphoramidite
chemistry as is known in the art. Standard phosphoramidite
chemistry involves the use of nucleosides comprising any of
5'-O-dimethoxytrityl, 2'-O-tert-butyldimethylsilyl,
3'-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and
exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4
acetyl cytidine, and N2-isobutyryl guanosine). Alternately,
2'-O-Silyl Ethers can be used in conjunction with acid-labile
2'-O-orthoester protecting groups in the synthesis of RNA as
described by Scaringe supra. Differing 2' chemistries can require
different protecting groups, for example 2'-deoxy-2'-amino
nucleosides can utilize N-phthaloyl protection as described by
Usman et al., U.S. Pat. No. 5,631,360, incorporated by reference
herein in its entirety).
[0635] During solid phase synthesis, each nucleotide is added
sequentially (3'- to 5'-direction) to the solid support-bound
oligonucleotide. The first nucleoside at the 3'-end of the chain is
covalently attached to a solid support (e.g., controlled pore glass
or polystyrene) using various linkers. The nucleotide precursor, a
ribonucleoside phosphoramidite, and activator are combined
resulting in the coupling of the second nucleoside phosphoramidite
onto the 5'-end of the first nucleoside. The support is then washed
and any unreacted 5'-hydroxyl groups are capped with a capping
reagent such as acetic anhydride to yield inactive 5'-acetyl
moieties. The trivalent phosphorus linkage is then oxidized to a
more stable phosphate linkage. At the end of the nucleotide
addition cycle, the 5'-O-protecting group is cleaved under suitable
conditions (e.g., acidic conditions for trityl-based groups and
Fluoride for silyl-based groups). The cycle is repeated for each
subsequent nucleotide.
[0636] Modification of synthesis conditions can be used to optimize
coupling efficiency, for example by using differing coupling times,
differing reagent/phosphoramidite concentrations, differing contact
times, differing solid supports and solid support linker
chemistries depending on the particular chemical composition of the
siNA to be synthesized. Deprotection and purification of the siNA
can be performed as is generally described in Usman et al., U.S.
Pat. No. 5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No.
6,437,117, and Bellon et al., U.S. Pat. No. 6,054,576, U.S. Pat.
No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringe supra,
incorporated by reference herein in their entireties. Additionally,
deprotection conditions can be modified to provide the best
possible yield and purity of siNA constructs. For example,
applicant has observed that oligonucleotides comprising
2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate
deprotection conditions. Such oligonucleotides are deprotected
using aqueous methylamine at about 35.degree. C. for 30 minutes. If
the 2'-deoxy-2'-fluoro containing oligonucleotide also comprises
ribonucleotides, after deprotection with aqueous methylamine at
about 35.degree. C. for 30 minutes, TEA-HF is added and the
reaction maintained at about 65.degree. C. for an additional 15
minutes. siNA molecules that are deprotected, purified, and/or
annealed are then formulated as described herein.
Example 5
RNAi In Vitro Assay to Assess siNA Activity
[0637] An in vitro assay that recapitulates RNAi in a cell-free
system is used to evaluate siNA constructs targeting RNA targets.
The assay comprises the system described by Tuschl et al., 1999,
Genes and Development, 13, 3191-3197 and Zamore et al., 2000, Cell,
101, 25-33 adapted for use with target RNA. A Drosophila extract
derived from syncytial blastoderm is used to reconstitute RNAi
activity in vitro. Target RNA is generated via in vitro
transcription from an appropriate hairless expressing plasmid using
T7 RNA polymerase or via chemical synthesis as described herein.
Sense and antisense siNA strands (for example 20 uM each) are
annealed by incubation in buffer (such as 100 mM potassium acetate,
30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at
90.degree. C. followed by 1 hour at 37.degree. C., then diluted in
lysis buffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH
at pH 7.4, 2 mM magnesium acetate). Annealing can be monitored by
gel electrophoresis on an agarose gel in TBE buffer and stained
with ethidium bromide. The Drosophila lysate is prepared using zero
to two-hour-old embryos from Oregon R flies collected on yeasted
molasses agar that are dechorionated and lysed. The lysate is
centrifuged and the supernatant isolated. The assay comprises a
reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM
final concentration), and 10% [vol/vol] lysis buffer containing
siNA (10 nM final concentration). The reaction mixture also
contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase,
100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL
RNasin (Promega), and 100 uM of each amino acid. The final
concentration of potassium acetate is adjusted to 100 mM. The
reactions are pre-assembled on ice and preincubated at 25.degree.
C. for 10 minutes before adding RNA, then incubated at 25.degree.
C. for an additional 60 minutes. Reactions are quenched with 4
volumes of 1.25.times.Passive Lysis Buffer (Promega). Target RNA
cleavage is assayed by RT-PCR analysis or other methods known in
the art and are compared to control reactions in which siNA is
omitted from the reaction.
[0638] Alternately, internally-labeled target RNA for the assay is
prepared by in vitro transcription in the presence of
[alpha-.sup.32P] CTP, passed over a G 50 Sephadex column by spin
chromatography and used as target RNA without further purification.
Optionally, target RNA is 5'-.sup.32P-end labeled using T4
polynucleotide kinase enzyme. Assays are performed as described
above and target RNA and the specific RNA cleavage products
generated by RNAi are visualized on an autoradiograph of a gel. The
percentage of cleavage is determined by PHOSPHOR IMAGER.RTM.
(autoradiography) quantitation of bands representing intact control
RNA or RNA from control reactions without siNA and the cleavage
products generated by the assay.
[0639] In one embodiment, this assay is used to determine target
sites the RNA target for siNA mediated RNAi cleavage, wherein a
plurality of siNA constructs are screened for RNAi mediated
cleavage of the RNA target, for example, by analyzing the assay
reaction by electrophoresis of labeled target RNA, or by northern
blotting, as well as by other methodology well known in the
art.
Example 6
Nucleic Acid Inhibition of Target RNA
[0640] siNA molecules targeted to the human target RNA are designed
and synthesized as described above. These nucleic acid molecules
can be tested for cleavage activity in vivo, for example, using the
following procedure.
[0641] Two formats are used to test the efficacy of siNAs targeting
target. First, the reagents are tested in cell culture to determine
the extent of RNA and protein inhibition. siNA reagents are
selected against the target as described herein. RNA inhibition is
measured after delivery of these reagents by a suitable
transfection agent to cells. Relative amounts of target RNA are
measured versus actin using real-time PCR monitoring of
amplification (e.g., ABI 7700 TAQMAN.RTM.). A comparison is made to
a mixture of oligonucleotide sequences made to unrelated targets or
to a randomized siNA control with the same overall length and
chemistry, but randomly substituted at each position. Primary and
secondary lead reagents are chosen for the target and optimization
performed. After an optimal transfection agent concentration is
chosen, a RNA time-course of inhibition is performed with the lead
siNA molecule. In addition, a cell-plating format can be used to
determine RNA inhibition.
Delivery of siNA to Cells
[0642] Cells are seeded, for example, at 1.times.10.sup.5 cells per
well of a six-well dish in EGM-2 (BioWhittaker) the day before
transfection. Formulated siNA compositions are complexed in EGM
basal media (Bio Whittaker) at 37.degree. C. for 30 minutes in
polystyrene tubes. Following vortexing, the complexed formulated
siNA composition is added to each well and incubated for the times
indicated. For initial optimization experiments, cells are seeded,
for example, at 1.times.10.sup.3 in 96 well plates and siNA complex
added as described. Efficiency of delivery of siNA to cells is
determined using a fluorescent siNA complexed with lipid. Cells in
6-well dishes are incubated with siNA for 24 hours, rinsed with PBS
and fixed in 2% paraformaldehyde for 15 minutes at room
temperature. Uptake of siNA is visualized using a fluorescent
microscope.
TAOMAN.RTM. (Real-Time PCR Monitoring of Amplification) and
Lightcycler Quantification of mRNA
[0643] Total RNA is prepared from cells following siNA delivery,
for example, using Qiagen RNA purification kits for 6-well or
Rneasy extraction kits for 96-well assays. For TAQMAN.RTM. analysis
(real-time PCR monitoring of amplification), dual-labeled probes
are synthesized with the reporter dye, FAM or JOE, covalently
linked at the 5'-end and the quencher dye TAMRA conjugated to the
3'-end. One-step RT-PCR amplifications are performed on, for
example, an ABI PRISM 7700 Sequence Detector using 50 .mu.l
reactions consisting of 10 .mu.l total RNA, 100 nM forward primer,
900 nM reverse primer, 100 nM probe, 1.times. TaqMan PCR reaction
buffer (PE-Applied Biosystems), 5.5 mM MgCl.sub.2, 300 .mu.M each
dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U
AMPLITAQ GOLD.RTM. (DNA polymerase) (PE-Applied Biosystems) and 10U
M-MLV Reverse Transcriptase (Promega). The thermal cycling
conditions can consist of 30 minutes at 48.degree. C., 10 minutes
at 95.degree. C., followed by 40 cycles of 15 seconds at 95.degree.
C. and 1 minute at 60.degree. C. Quantitation of mRNA levels is
determined relative to standards generated from serially diluted
total cellular RNA (300, 100, 33, 11 ng/rxn) and normalizing to
.beta.-actin or GAPDH mRNA in parallel TAQMAN.RTM. reactions
(real-time PCR monitoring of amplification). For each gene of
interest an upper and lower primer and a fluorescently labeled
probe are designed. Real time incorporation of SYBR Green I dye
into a specific PCR product can be measured in glass capillary
tubes using a lightcyler. A standard curve is generated for each
primer pair using control cRNA. Values are represented as relative
expression to GAPDH in each sample.
Western Blotting
[0644] Nuclear extracts can be prepared using a standard micro
preparation technique (see for example Andrews and Faller, 1991,
Nucleic Acids Research, 19, 2499). Protein extracts from
supernatants are prepared, for example using TCA precipitation. An
equal volume of 20% TCA is added to the cell supernatant, incubated
on ice for 1 hour and pelleted by centrifugation for 5 minutes.
Pellets are washed in acetone, dried and resuspended in water.
Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear
extracts) or 4-12% Tris-Glycine (supernatant extracts)
polyacrylamide gel and transferred onto nitro-cellulose membranes.
Non-specific binding can be blocked by incubation, for example,
with 5% non-fat milk for 1 hour followed by primary antibody for 16
hour at 4.degree. C. Following washes, the secondary antibody is
applied, for example (1:10,000 dilution) for 1 hour at room
temperature and the signal detected with SuperSignal reagent
(Pierce).
Example 7
Evaluation of Serum Stability of Formulated siNA Compositions
[0645] As discussed herein, one way to determine the transfection
or delivery efficiency of the formulated lipid composition is to
determine the stability of the formulated composition in serum in
vitro. Relative turbity measurement can be used to determine the in
vitro serum stability of the formulated siNA compositions.
[0646] Turbidity measurements were employed to monitor the serum
stability of lipid particle formulations L065, F2, L051, and L073
(see FIGS. 8 and 9 for the lipid formulations of L051 and L073).
The lipid formulation of L065 comprises cationic lipid CpLinDMA,
neutral lipid DSPC, cholesterol, and 2kPEG-DMG. The lipid
formulation F2 comprises DODAP. The absorbance of formulated siNA
compositions (0.1 mg/ml) in the absence and presence of 50% serum
was measured at 500 nm with a corresponding amount of serum alone
as a reference by using SpectraMax.RTM. Plus384 microplate
spectrophotometer from Molecular Devices (Sunnyvale, Calif.). The
formulations were incubated at 37.degree. C. and analyzed at 2 min,
5 min, 10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 7 h and 24
h. Relative turbidity was determined by dividing the sample
turbidity by the turbidity of 2 min formulated siNA compositions
incubated in 50% serum. A formulated molecular composition is
stable in serum if the relative turbidity remains constant around
1.0 over time. As shown in FIG. 11, formulated siNA compositions
L065, L051, and L073 are serum-stable lipid nanoparticle
compositions. As shown in FIG. 33, formulated siNA compositions
L077, L080, L082 and L083, are serum-stable lipid nanoparticle
compositions.
Example 8
Evaluation of pH-Dependent Phase Transition of Formulated siNA
Compositions
[0647] Additionally, the transfection or delivery efficiency of the
formulated lipid composition can be determined by determining the
pH-dependent phase transition of the formulated composition in
vitro. Relative turbity measurement can be used to determine the
pH-dependent phase transition of formulated siNA compositions in
vitro.
[0648] Turbidity measurement was employed to monitor the phase
transition of formulated siNA compositions L065, L051, F2, L073,
and L069. The absorbance of lipid particle formulations (0.1 mg/ml)
in 0.1 M phosphate buffer with pH at 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,
6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 was measured at 350 nm with a
corresponding amount of buffer alone as a reference by using
SpectraMax.RTM. Plus384 microplate spectrophotometer from Molecular
Devices (Sunnyvale, Calif.). This assay measures the relative light
scattering of the formulations at various pH. The lamellar
structure (i.e., serum stable structure) having realatively bigger
particle size is expected to scatter more light than the
corresponding inverted hexagonal structure. The samples were
incubated at 37.degree. C. and analyzed at 2 min, 5 min, 10 min, 30
min, and 2 h. Relative turbidity was determined by dividing the
sample turbidity by the turbidity of 2 min formulated siNA
compositions incubated in phosphate buffer at pH 7.5. A formulated
molecular composition undergoes pH-dependent phase transition if
there is a change in the relative turbidity when measured between
pH 7.5-pH 5.0. As shown in FIG. 12, formulated siNA compositions
L051 and L073 undergo pH-dependent phase transition at pH 6.5-pH
5.0. As shown in FIG. 13, formulated siNA composition L069
undergoes pH-dependent phase transition at pH 6.5-pH 5.0. As shown
in FIG. 34, formulated siNA compositions L077, L080, L082, and L083
undergo pH-dependent phase transition at pH 6.5-pH 5.0.
Example 9
Evaluation of Formulated siNA Compositions in Models of Chronic HBV
Infection
[0649] In Vitro Analysis of siNA Nanoparticle Activity
[0650] Hep G2 cells were grown in EMEM (Cellgro Cat #10-010-CV)
with non-essential amino acids, sodium pyruvate (90%), and 10%
fetal bovine serum (HyClone Cat #SH30070.03). Replication competent
cDNA was generated by the excision and re-ligation of the HBV
genomic sequences from the psHBV-1 vector. HepG2 cells were plated
(3.times.10.sup.4 cells/well) in 96-well microtiter plates and
incubated overnight. A cationic lipid/DNA complex was formed
containing (at final concentrations) cationic lipid (11-15
.mu.g/mL), and re-ligated psHBV-1 (4.5 .mu.g/mL) in growth media.
Following a 15 min incubation at 37.degree. C., 20 .mu.L of the
complex was added to the plated HepG2 cells in 80 .mu.L of growth
media minus antibiotics. After 7.5 hours at 37.degree. C., the
media was then removed, the cells rinsed once with media, and 100
.mu.L of fresh media was added to each well. 50 .mu.L of the siNA
nanoparticle formulation (see Example 9 for formulation details)
(diluted into media at a 3.times. concentration) was added per
well, with 3 replicate wells per concentration. The cells were
incubated for 4 days, the media was then removed, and assayed for
HBsAg levels. FIG. 15 shows level of HBsAg from formulated
(Formulation L051, Table IV) active siNA treated cells compared to
untreated or negative control treated cells. FIG. 16 shows level of
HBsAg from formulated (Formulations L053 and L054, Table IV) active
siNA treated cells compared to untreated or negative control
treated cells. FIG. 17 shows level of HBsAg from formulated
(Formulation L051, Table IV) active siNA treated cells compared to
untreated or negative control treated cells. FIG. 30 shows level of
HBsAg from formulated (Formulations L083 and L084, Table IV) active
siNA treated cells compared to untreated or negative control
treated cells. FIG. 31 shows level of HBsAg from formulated
(Formulation L077, Table IV) active siNA treated cells compared to
untreated or negative control treated cells. FIG. 32 shows level of
HBsAg from formulated (Formulation L080, Table IV) active siNA
treated cells compared to untreated or negative control treated
cells. In these studies, a dose dependent reduction in HBsAg levels
was observed in the active formulated siNA treated cells using
nanoparticle formulations L051, L053, and L054, while no reduction
is observed in the negative control treated cells. This result
indicates that the formulated siNA compositions are able to enter
the cells, and effectively engage the cellular RNAi machinery to
inhibit viral gene expression.
Analysis of Formulated siRNA Activity in a Mouse Model of HBV
Replication
[0651] To assess the activity of chemically stabilized siNA
nanoparticle (see Example 9 for formulation details) compositions
against HBV, systemic dosing of the formulated siNA composition
(Formulation L051, Table IV) was performed following hydrodynamic
injection (HDI) of the HBV vector in mouse strain
NOD.CB17-Prkdc.sup.scid/J (Jackson Laboratory, Bar Harbor, Me.).
Female mice were 5-6 weeks of age and approximately 20 grams at the
time of the study. The HBV vector used, pWTD, is a head-to-tail
dimer of the complete HBV genome. For a 20-gram mouse, a total
injection of 1.6 ml containing pWTD in saline, was injected into
the tail vein within 5 seconds. A total of 0.3 .mu.g of the HBV
vector was injected per mouse. In order to allow recovery of the
liver from the disruption caused by HDI, dosing of the formulated
siNA compositions were started 6 days post-HDI. Encapsulated active
or negative control siRNA were administered at 3 mg/kg/day for
three days via standard IV injection. Groups (N=5) of animals were
sacrificed at 3 and 7 days following the last dose, and the levels
of serum HBV DNA and HBsAg were measured. HBV DNA titers were
determined by quantitative real-time PCR and expressed as mean log
10 copies/ml (.+-.SEM). The serum HBsAg levels were assayed by
ELISA and expressed as mean log 10 pg/ml (.+-.SEM). Significant
reductions in serum HBV DNA (FIGS. 18 and 29) and HBsAg (FIGS. 19,
30, 31, and 32)) were observed at both the 3 and 7-day time points
in the active formulated siNA composition treated groups as
compared to both the PBS and negative control groups.
Materials and Methods
Oligonucleotide Synthesis and Characterization
[0652] All RNAs were synthesized as described herein. Complementary
strands were annealed in PBS, desalted and lyophilized. The
sequences of the active site 263 HBV siNAs are shown in FIG. 14.
The modified siNAs used in vivo are termed HBV263M and HBV1583M,
while versions containing unmodified ribonucleotides and inverted
abasic terminal caps are called HBV263R and HBV1583R. Some
pharmacokinetic studies were done with siNA targeting two other
sites, HBV1580M and HBV1580R.
[0653] The siNA sequences for HCV irrelevant control are:
TABLE-US-00001 sense strand: 5' B-cuGAuAGGGuGcuuGcGAGTT-B 3' (SEQ
ID NO: 1) antisense strand: 5' CUCGcAAGcAcccuAucAGTsT 3' (SEQ ID
NO: 2)
[0654] (where lower case=2'-deoxy-2'-flouro; Upper Case
italic=2'-deoxy; Upper Case underline=2'-O-methyl; Upper Case
Bold=ribonucleotide; T=thymidine; B=inverted deoxyabasic; and
s=phosphorothioate)
[0655] The inverted control sequences are inverted from 5' to
3'.
HBsAg ELISA Assay
[0656] Levels of HBsAg were determined using the Genetic
Systems/Bio-Rad (Richmond, Va.) HBsAg ELISA kit, as per the
manufacturer's instructions. The absorbance of cells not
transfected with the HBV vector was used as background for the
assay, and thus subtracted from the experimental sample values.
HBV DNA Analysis
[0657] Viral DNA was extracted from 50 .mu.L mouse serum using
QIAmp 96 DNA Blood kit (Qiagen, Valencia, Calif.), according to
manufacture's instructions. HBV DNA levels were analyzed using an
ABI Prism 7000 sequence detector (Applied Biosystems, Foster City,
Calif.). Quantitative real time PCR was carried out using the
following primer and probe sequences: forward primer
5'-CCTGTATTCCCATCCCATCGT (SEQ ID NO: 3, HBV nucleotide 2006-2026),
reverse primer 5'-TGAGCCAAGAGAAACGGACTG (SEQ ID NO: 4, HBV
nucleotide 2063-2083) and probe FAM 5'-TTCGCA AAATACCTATGGGAGTGGGCC
(SEQ ID NO: 5, HBV nucleotide 2035-2062). The psHBV-1 vector,
containing the full length HBV genome, was used as a standard curve
to calculate HBV copies per mL of serum.
Example 10
Evaluation of Formulated siNA Compositions in an In Vitro HCV
Replicon Model of HCV Infection
[0658] An HCV replicon system was used to test the efficacy of
siNAs targeting HCV RNA. The reagents were tested in cell culture
using Huh7 cells (see for example Randall et al., 2003, PNAS USA,
100, 235-240) to determine the extent of RNA inhbition. siNA were
selected against the HCV target as described herein. The active
siNA sequences for HCV site 304 are as follows: sense strand: (SEQ
ID NO: 1); antisense strand: (SEQ ID NO: 2) (these were used as
inactive sequences in Example 8 above). The siNA inactive control
sequences used in the study target HBV site 263 and are as follows:
sense strand: (SEQ ID NO: 6); antisense strand: (SEQ ID NO: 7),
(these were used as active sequences in Example 8 above). The
active and inactive siNAs were formulated as Formulation L051,
L053, or L054 as described in Example 9 above. Huh7 cells,
containing the stably transfected Clone A HCV subgenomic replicon
(Apath, LLC, St. Louis, Mo.), were grown in DMEM (Invitrogen
catalog #11965-118) with 5 mls of 100.times. (10 mM) Non-Essential
Amino Acids (Invitrogen catalog #11140-050), 5 uL of 200 mM
Glutamine (Cellgro catalog #25-005-C1), 50 uL of heat inactivated
Fetal Bovine Serum (Invitrogen catalog #26140-079) and 1 mg/mLG418
(Invitrogen catalog #11811-023). For transfection with siNA
formulations, cells are plated at 9,800 cells per well into a
96-well CoStar tissue culture plate using DMEM with NEAA and 10%
FBS, (no antibiotics). After 20-24 hours, cells were transfected
with formulated siNA for a final concentration of 1-25 nM. After
incubating for 3 days, the cells were lysed and RNA extracted using
the RNaqueous-96 kit (Ambion Cat #1920) as per the manufacturers
instructions. FIG. 20 shows level of HCV RNA from formulated
(Formulation L051, Table IV) active siNA treated cells compared to
untreated or negative control treated cells. FIG. 21 shows level of
HCV RNA from formulated (Formulations L053 and L054, Table IV)
active siNA treated cells compared to untreated or negative control
treated cells. In these studies, a dose dependent reduction in HCV
RNA levels was observed in the active formulated siNA treated cells
using formulations L051, L053, and L054, while no reduction is
observed in the negative control treated cells. This result
indicates that the formulated siNA compositions are able to enter
the cells, and effectively inhibit viral gene expression.
Example 11
Lung Distribution of Unformulated and Formulated siNA After
Intratracheal Dosing
[0659] To determine the efficiency of delivery of siNA molecules to
the lung, unformulated siRNA (naked), cholesterol conjugated siNA,
or siRNA in formulated molecular compositions (T018.1 and T019.1)
were administered via the trachea to the lungs of mice.
Unformulated siNA comprises naked nucleic acid. Cholesterol
conjugated siNA comprises siNA linked to cholesterol. Formulated
molecular compositions T018.1 and T019.1 comprise siNA formulated
with DOcarbDAP, DSPC, cholesterol and PEG-DMG, and DODMA, DSPC,
cholesterol and PEG-DMG, respectively. Groups of three female C57
B1/6 mice were placed under anesthesia with ketamine and xylazine.
Filtered dosing solutions were administered via the trachea at 1.0
mg/kg duplexed siRNA, using a Penncentury model #1A-1C microsprayer
and a Penncentury model #FMJ250 syringe to aerosolize the siRNA
(TGF.beta. site 1264 stabilization chemistry 7/8) directly into the
lungs. Animals were dosed with unformulated siNA,
cholesterol-conjugated siNA or siNA in formulated molecular
compositions. At 1, 24 or 72 hours after dosing, the animals were
euthanized, exsanguinated and perfused with sterile veterinary
grade saline via the heart. The lungs were removed, placed in a
pre-weighed homogenization tube and frozen on dry ice. Lung weights
were determined by subtraction after weighing the tubes plus lungs.
Levels of siNA in the lung tissue were determined using a
hybridization assay. FIG. 22, shows the levels of siNA in lung
tissue after direct dosing of (i) unformulated siNA, (ii)
cholesterol conjugated siNA or (iii) siNA in formulated molecular
compositions T018.1 or T019.1. Half lives of exposure in lung
tissue were 3-4 hours for the unformulated siNA, 9 hours for the
cholesterol conjugated siNA and 37-39 hours for the siNA in
formulated molecular compositions TO 18.1 or TO 19.1.
Example 12
Efficient Transfection of Various Cell Lines Using siNA LNP
Formulations of the Invention
[0660] The transfection efficacy of LNP formuations of the
invention was determined in various cell lines, including 6.12
spleen, Raw 264.7 tumor, MM14Lu, NIH 3T3, D10.G4.1 Th2 helper, and
lung primary macrophage cells by targeting endogenous MAP Kinase 14
(p38) gene expression. A potent lead siNA against MapK14 (p38a) was
selected by in vitro screening using Lipofectamine 2000 (LF2K) as
the delivery agent. The sense strand sequence of this siNA
comprised 5'-B cuGGuAcAGAccAuAuuGATT B-3' (SEQ ID NO: 6) and the
antisense strand sequence comprised 5'-UCAAuAuGGucuGuAccAGTsT-3'
(SEQ ID NO: 7), where lower case=2'-deoxy-2'-flouro; Upper Case
italic=2'-deoxy; Upper Case underline=2'-O-methyl; Upper Case
Bold=ribonucleotide; T=thymidine; B=inverted deoxyabasic; and
s=phosphorothioate).
[0661] Proprietary MapK14 targeted LNPs were screened and compared
to LF2K and a LNP control containing an inactive siNA in cultured
cells. Furthermore, lead LNPs were tested in a dose response method
to determine IC50 values. Results are summarized in Table V. FIG.
35 shows efficacy data for LNP 58 and LNP 98 formulations targeting
MapK14 site 1033 in RAW 264.7 mouse macrophage cells. FIG. 36 shows
efficacy data for LNP 98 formulations targeting MapK14 site 1033 in
MM14.Lu normal mouse lung cells. FIG. 37 shows efficacy data for
LNP 54, LNP 97, and LNP 98 formulations targeting MapK14 site 1033
in 6.12 B lymphocyte cells. FIG. 38 shows efficacy data for LNP 98
formulations targeting MapK14 site 1033 in NIH 3T3 cells. FIG. 39
shows the dose-dependent reduction of MapK14 RNA via MapK14 LNP 54
and LNP 98 formulated siNAs in RAW 264.7 cells. FIG. 40 shows the
dose-dependent reduction of MapK14 RNA via MapK14 LNP 98 formulated
siNAs in MM14.Lu cells. FIG. 41 shows the dose-dependent reduction
of MapK14 RNA via MapK14 LNP 97 and LNP 98 formulated siNAs in 6.12
B cells. FIG. 42 shows the dose-dependent reduction of MapK14 RNA
via MapK14 LNP 98 formulated siNAs in NIH 3T3 cells.
LF2K Transfection Method.
[0662] The following procecure was used for LF2K transfection.
After 20-24 hours, cells were transfected using 0.25 or 0.35 uL
Lipofectamine 2000/well and 0.15 or 0.25 uL/well, complexed with 25
nM siNA. Lipofectamine 2000 was mixed with OptiMEM and allowed to
sit for at least 5 minutes. For 0.25 uL transfections, 1 uL of LF2K
was mixed with 99 uL OptiMEM for each complex. For 0.35 uL
transfections, 1.4 uL of LF2K was mixed with 98.6 uL OptiMEM for
each complex. For 0.15 uL transfections, 0.60 uL of SilentFect was
mixed with 99.4 uL OptiMEM for each complex . For 0.30 uL
transfections, 1.2 uL of SilentFect was mixed with 98.2 uL OptiMEM
for each complex. The siNA was added to a microtitre tube (BioRad
#223-9395) and OptiMEM was then added to make 100 uL total volume
to be used in 4 wells. 100 uL of the Lipofectamine 2000/OptiMEM
mixture was added and the tube was vortexed on medium speed for 10
seconds and allowed to sit at room temperature for 20 minutes. The
tube was vortexed quickly and 50uL was added per well, which
contained 100 uL media. RNA from treated cells was isolated at 24,
48, 72, and 96 hours.
LNP Transfection Method:
[0663] The following procecure was used for LF2K transfection.
Cells were plated to the desired concentration in 100 uL of
complete growth medium in 96-well plates, ranging from from
5,000-30,000 cells/well. After 24 hours, the cells were transfected
by diluting a 5.times. concentration of LNP in complete growth
medium onto the cells, (25 uL of 5.times. results in a final
concentration of 1.times.). RNA from treated cells was isolated at
24, 48, 72, and 96 hours.
Example 13
Reduction of Airway Hyper-Responsiveness in a Mouse Model of
Asthma
[0664] An OVA induced airway hyper-responsiveness model was used to
evaluate LNP formulated siNA molecules targeting interleukin 4R
(IL-4R alpha) for efficacy in reducing airway hyper-responsiveness.
The sense strand sequence of the active siNA targeting IL-4R alpha
used in this study comprised 5'-B ucAGcAuuAccAAGAuuAATT B-3' (SEQ
ID NO: 8) and the antisense strand sequence comprised
5'-UUAAucuuGGuAAuGcuGATsT-3' (SEQ ID NO: 9), where lower
case=2'-deoxy-2'-flouro; Upper Case italic=2'-deoxy; Upper Case
underline=2'-O-methyl; Upper Case Bold=ribonucleotide; T=thymidine;
B=inverted deoxyabasic; and s=phosphorothioate). On Day 0 and 7,
the animals were immunized by intraperitoneal injection of 0.4
mg/mL OVA/saline solution mixed in an equal volume of Imject Alum
for a final injection solution of 0.2 mg/mL (100 uL/mouse). LNP-51
formulated IL-4R-alpha Site 1111 siNA (see U.S. Ser. No.
11/001,347, incorporated by reference herein), prepared in PBS (w/o
Ca2+, Mg2+), or irrelevant control was delivered by intratracheal
dosing qd (once every day) beginning on Day 17 and ending on Day 26
for a total of 10 doses. Mice were aerosol challenged with OVA
(1.5% in saline) for 30 minutes on days 24, 25 and 26 using the
Pari LC aerosol nebulizer. Animals were allowed to rest for 24
hours prior to airway function analysis. On Day 28 airway
responsiveness was assessed after challenge with aerosolized
methacholine using the Buxco Whole Body Plethysmograph. After
methacholine challenge, animals were euthanized. A tracheotomy was
performed, and the lungs were lavaged with 0.5 mL of saline twice.
Lung lavage was performed while massaging the animal's chest and
all lavage fluid were collected and placed on ice. A cytospin
preparation was performed to collect the cells from the BAL fluid
for differential cell counts. Results are shown in FIG. 43, which
clearly demonstrates the activity of the formulated siNA in a dose
response (0.01, 0.1, and 1 mg/kg) compared to the LNP vehicle alone
and untreated (naive) animals.
Example 14
Efficient Reduction in Human Huntingtin (htt) Gene Expression In
Vivo Using LNP Formulated siNA
[0665] Huntington's disease (HD) is a dominant neurodegenerative
disorder caused by an expansion in the polyglutamine (polyQ) tract
of the huntingtin (htt) protein. PolyQ expansion in htt induces
cortical and striatal neuron cell less, and the formation of
htt-containing aggregates within brain cells. HD patients have
progressive psychiatric, cognitive and motor dysfunction and
premature death. Early work in mouse models has demonstrated that
reduction of mutant protein after the onset of disease phenotypes
could improve motor dysfunction and reduce htt-aggregate burden.
Thus, reduction of mutant htt in patient brain may improve the
disease.
[0666] Recent work has shown that reduction of mutant htt in a
mouse model of HD, using a viral vector expressing short
interfering RNAs (siRNAs), protected the animal from the onset of
behavioral and neuropathological hallmarks of the disease (see
Harper et al., 2005, PNAS USA, 102: 5820-5). This study was
utilized to determine whether delivery of synthetic siNAs directly
to the brain by nonviral methods could be similarly effective. This
approach has many advantages, including the ability to modify
dosing regimines. Chemically modified siNA, sense strand having
sequence 5'-B AccGuGuGAAucAuuGucuTT B-3' (SEQ ID NO:10) and
antisense strand 5'-AGAcAAuGAuucAcAcGGuTsT-3' (SEQ ID NO:11)
encapsulated in lipid nanoparticles (LNP) formulations LNP-061,
LNP-098, and LNP-101 (see Table IV) were utilized in this study. In
these sequences, lower case stands for 2'-deoxy-2'-fluoro, Upper
Case stands for ribonucleotides, underline Upper Case stands for
2'-O-methyl nucleotides, T is thymidine, s is phosphorothioate, and
B is inverted deoxy abasic. The siNA duplexes encapsulated in the
various LNP formulations were screened for their ability to silence
full-length htt in vitro, followed by testing in vivo. Using Alzet
osmotic pumps, siNAs encapsulated in LNPs were infused into the
lateral ventrical or striatum for 7 or 14. days, respectively, at
concentrations ranging from 0.1 to 1 mg/ml (total dose ranging from
8.4 to 84 .mu.g). An impressive 80% reduction in htt mRNA levels
was observed in animals treated with LNP-061 and LNP-098 formulated
siNA as determined by QPCR compared to scrambled control sequences,
or naive brain. Results are shown in FIG. 44.
Example 14
Preparation of Cationic Lipids of the Invention (see FIGS. 23A and
23B for Synthetic Schemes)
Cholest-5-en-3.beta.-tosylate (2)
[0667] Cholesterol (1, 25.0 g, 64.7 mmol) was weighed into a 1 L
round bottomed flask with a stir bar. The flask was charged with
pyridine (250 mL), septum sealed and flushed with argon.
Toluenesulfonyl chloride (25.0 g, 131 mmol) was weighed into a 100
mL round bottomed flask, which was then sealed and charged with
pyridine. The toluenesulfonyl chloride solution was then
transferred, via syringe, to the stirring cholesterol solution,
which was allowed to stir overnight. The bulk of pyridine was
removed in vacuo and the resulting solids were suspended in
methanol (300 mL) and stirred for 3 hours, until the solids were
broken up into a uniform suspension. The resultant suspension was
filtered and the solids were washed with acetonitrile and dried
under high vacuum to afford 31.8 g (91%) of a white powder (see for
example Davis, S. C.; Szoka, F. C., Jr. Bioconjugate Chem. 1998, 9,
783).
Cholest-5-en-3.beta.-oxybutan-4-ol (3a)
[0668] Cholest-5-ene-3.beta.-tosylate (20.0 g, 37.0 mmol) was
weighed into a 500 mL round bottomed flask with a stir bar. The
flask was charged with dioxane (300 mL) and 1,4-butanediol (65.7
mL, 20 equiv.). The flask was fitted with a reflux condenser and
the mixture was brought to reflux overnight. The reaction was
cooled and concentrated in vacuo. The reaction mixture was
suspended in water (400 mL). The solution was extracted with
methylene chloride (3.times.200 mL). The organic phases were
combined and washed with water (2.times.200), dried over magnesium
sulfate, filtered and the solvent removed. The resultant oil/wax
was further purified via column chromatography (15%
Acetone/Hexanes) to afford 13.41 g (79%) of a colorless wax.
Cholest-5-en-3.beta.-oxypent-3-oxa-an-5-ol (3b)
[0669] This compound was prepared similarly to
cholest-5-en-3.beta.-oxybutan-4-ol. Cholest-5-ene-3.beta.-tosylate
(5.0 g, 9.2 mmol) was weighed into a 500 mL round bottomed flask
with a stir bar. The flask was charged with dioxane (150 mL) and
diethylene glycol (22 mL, 25 equiv.). The flask was fitted with a
reflux condenser and the mixture was brought to reflux overnight.
The reaction was cooled and concentrated. The reaction mixture was
suspended in water (500 mL). The solution was extracted with
methylene chloride (3.times.200 mL). The organic phases were
combined and washed with water (2.times.200 mL), dried over
magnesium sulfate, filtered and the solvent removed. The resultant
oil/wax was further purified via column chromatography (25%
EtOAc/Hexanes) to afford 3.60 g (82%) of colorless oil (see for
example Davis, S. C.; Szoka, F. C., Jr. Bioconjugate Chem. 1998, 9,
783).
Cholest-5-en-3.beta.-oxybutan-4-mesylate (4a)
[0670] Cholest-5-en-3.beta.-oxybutan-4-ol (12.45 g, 27.14 mmol) was
weighed into a 500 mL round bottomed flask with a stir bar. The
flask was sealed, flushed with argon, charged with methylene
chloride (100 mL) and triethylamine (5.67 mL, 1.5 equiv.) and
cooled to 0.degree. C. Methanesulfonyl chloride (3.15 mL, 1.5
equiv.) was measured in a PP syringe and added slowly to the
stirring reaction mixture. The reaction was allowed to stir for 1
hr at 0.degree. C. when TLC analysis (7.5% EtOAc/Hexanes) showed
that the reaction was complete. The reaction mixture was diluted
with methylene chloride (100 mL) and washed with saturated
bicarbonate solution (2.times.200 mL) and brine (1.times.100 mL).
The organic phase was dried over MgSO.sub.4, filtered and
concentrated to give 14.45 g (99%) of a colorless wax that was used
without further purification.
Cholest-5-en-3.beta.-oxypent-3-oxa-an-5-mesylate (4b)
[0671] This compound was prepared similarly to
Cholest-5-en-3.beta.-oxybutan-4-mesylate.
Cholest-5-en-3.beta.-oxypent-3-oxa-an-5-ol (3.60 g, 7.58 mmol) was
weighed into a 500 mL round bottomed flask with a stir bar. The
flask was sealed, flushed with argon, charged with methylene
chloride (30 mL) and triethylamine (1.60 mL, 1.5 equiv.) and cooled
to 0.degree. C. Methanesulfonyl chloride (0.89 mL, 1.5 equiv.) was
measured in a PP syringe and added slowly to the stirring reaction
mixture. The reaction was allowed to stir for 1 hr at 0.degree. C.
when TLC analysis (10% EtOAc/Hexanes) showed that the reaction was
complete. The reaction mixture was diluted with methylene chloride
(150 mL) and washed with saturated bicarbonate solution
(2.times.100 mL) and brine (1.times.100 mL). The organic phase was
dried over MgSO.sub.4, filtered and concentrated to give 4.15 g
(99%) of a colorless wax that was used without further
purification.
1-(4,4'-Dimethoxytrityloxy)-3-dimethylamino-2-propanol (5)
[0672] 3-Dimethylamino-1,2-propanediol (6.0 g, 50 mmol) was weighed
into a 1 L round bottomed flask with a stir bar. The flask was
sealed, flushed with argon, charged with pyridine and cooled to
0.degree. C. 4,4'-Dimethoxytrityl chloride (17.9 g, 1.05 equiv.)
was weighed into a 100 mL round bottomed flask, sealed and then
dissolved in pyridine (80 mL). The 4,4'-dimethoxytrityl chloride
solution was transferred to the stirring reaction mixture slowly,
using additional fresh pyridine (20 mL) to effect the transfer of
residual 4,4'-dimethoxytrityl chloride. The reaction was allowed to
come to room temperature while stirring overnight. The reaction was
concentrated in vacuo and re-dissolved in dichloromethane (300 mL).
The organic phase was washed with saturated bicarbonate
(2.times.200 mL) and brine (1.times.200 mL), dried over MgSO.sub.4,
filtered, concentrated and dried under high vacuum to afford 22.19
g of a yellow gum that was used without further purification.
3-Dimethylamino-2-(cholest-5-en-3.beta.-oxybutan-4-oxy)-1-propanol
(6a)
[0673] 1-(4,4'-Dimethoxytrityloxy)-3-Dimethylamino-2-propanol (7.50
g, 17.8 mmol) was weighed into a 200 mL round bottomed flask and
co-evaporated with anhydrous toluene (2.times.50 mL). A stir bar
was added to the flask which was septum sealed, flushed with argon
and charged with toluene (60 mL). Sodium hydride (1.71 g, 4 equiv.)
was added at once and the mixture was stirred at room temperature
for 20 minutes. Cholest-5-en-3.beta.-oxybutan-4-mesylate was
dissolved in anhydrous toluene (20 mL) and added to the reaction
mixture, via syringe. The flask was fitted with a reflux condenser
with a continuous argon stream and the reaction was heated to
reflux overnight. The reaction mixture was cooled to room
temperature in a water bath and ethanol was added dropwise until
gas evolution ceased. The reaction mixture was diluted with ethyl
acetate (300 mL) and washed with aqueous 10% sodium carbonate
(2.times.300 mL). The aqueous phases were combined and back
extracted with ethyl acetate (2.times.100 mL). The organic phases
were combined, dried over MgSO.sub.4, filtered and concentrated to
an oil in a 500 mL round bottomed flask.
[0674] The flask was fitted with a stir bar, sealed, purged with
argon and charged with dichloroacetic acid solution (3% in DCM, 200
mL). Triethylsilane (14.2 mL, 89 mmol) was added to the mixture and
the reaction was allowed to stir overnight. The reaction mixture
was diluted with DCM (300 mL) and washed with saturated bicarbonate
solution (2.times.200 mL). The aqueous phases were combined and
back extracted with DCM (2.times.100 mL). The organic phases were
combined and dried over MgSO.sub.4, filtered and concentrated to an
oil that was re-dissolved in ethanol (150 mL). Potassium fluoride
(10.3 g, 178 mmol) was added to the solution, which was then
brought to reflux for 1 hr. The mixture was cooled, concentrated in
vacuo, re-dissolved in DCM (200 mL), filtered and concentrated to
an oil/crystal mixture. The mixture was re-dissolved in a minimum
of DCM and loaded onto a silica gel column which was
pre-equilibrated and eluted with 25% EtOAc/Hexanes with 3% TEA to
afford 4.89 g (49%) of a colorless wax.
3-Dimethylamino-2-(cholest-5-en-3.beta.-oxypent-3-oxa-an-5-oxy)-1-propanol
(6b)
[0675] This compound was prepared similarly to
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-propanol.
1-(4,4'-Dimethoxytrityloxy)-3-Dimethylamino-2-propanol (2.65 g,
6.31 mmol) was weighed into a 200 mL round bottomed flask and
co-evaporated with anhydrous toluene (2.times.20 mL). A stir bar
was added to the flask which was septum sealed, flushed with argon
and charged with toluene (50 mL). Sodium hydride (0.61 g, 4 equiv.)
was added at once and the mixture was stirred at room temperature
for 20 minutes. Cholest-5-en-3.beta.-oxypent-3-oxa-an-5-mesylate
(4.15 g, 7.6 mmol) was dissolved in anhydrous toluene (10 mL) and
added to the reaction mixture, via syringe. The flask was fitted
with a reflux condenser with a continuous argon stream and the
reaction was heated to reflux overnight. The reaction mixture was
cooled to room temperature in a water bath and ethanol was added
dropwise until gas evolution ceased. The reaction mixture was
diluted with ethyl acetate (200 mL) and washed with aqueous 10%
sodium carbonate (2.times.200 mL). The aqueous phases were combined
and back extracted with ethyl acetate (2.times.100 mL). The organic
phases were combined, dried over MgSO.sub.4, filtered and
concentrated to an oil in a 500 mL round bottomed flask.
[0676] The flask was fitted with a stir bar, sealed, purged with
argon and charged with dichloroacetic acid solution (3% in DCM, 150
mL). Triethylsilane (4.03 mL, 25.2 mmol) was added to the mixture
and the reaction was allowed to stir for 4 hours. The reaction
mixture was diluted with DCM (100 mL) and washed with saturated
bicarbonate solution (2.times.200 mL). The aqueous phases were
combined and back extracted with DCM (2.times.100 mL). The organic
phases were combined and dried over MgSO.sub.4, filtered and
concentrated to an oil that was re-dissolved in ethanol (100 mL).
Potassium fluoride (3.6 g, 63 mmol) was added to the solution,
which was then brought to reflux for 1 hr. The mixture was cooled,
concentrated in vacuo, re-dissolved in DCM (200 mL), filtered and
concentrated to an oil/crystal mixture. The mixture was
re-dissolved in a minimum of DCM and loaded onto a silica gel
column which was pre-equilibrated and eluted with 25%
Acetone/Hexanes with 3% TEA to afford 2.70 g (74%) of a colorless
wax.
Linoleyl mesylate (7)
[0677] Linoleyl alcohol (10.0 g, 37.5 mmol) was weighed into a 500
mL round bottomed flask with a stir bar. The flask was sealed,
flushed with argon, charged with DCM (100 mL) and triethylamine
(7.84 mL, 1.5 equiv.) and cooled to 0.degree. C. Methanesulfonyl
chloride (4.35 mL), 1.5 equiv.) was measured in a PP syringe and
added slowly to the stirring reaction mixture. TLC analysis (7.5%
EtOAc/Hexanes) showed the reaction was complete within 1 hr. The
reaction was diluted with DCM (100 mL) and washed with saturated
bicarbonate solution (2.times.200 mL). The organic phase was dried
over MgSO.sub.4, filtered and concentrated to give 12.53 g (97%) of
colorless oil that was used without further purification.
3-Dimethylamino-2-(cholest-5-en-3.beta.-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (8a) (CLinDMA)
[0678]
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-propanol
(2.6 g, 4.6 mmol) was weighed into a 200 mL round bottomed flask
and co-evaporated with anhydrous toluene 2.times.20 mL). A stir bar
was added to the flask, which was then sealed, flushed with argon
and charged with anhydrous toluene (100 mL). Sodium hydride (0.7 g,
6 equiv) was added at once and the mixture was stirred, under
argon, for 20 minutes. Linoleyl mesylate (4.6 g, 2.3 equiv.) was
measured in a PP syringe and added slowly to the reaction mixture.
The flask was fitted with a reflux condenser and the apparatus was
flushed with argon. The reaction mixture was heated in an oil bath
and allowed to stir at reflux overnight. The reaction mixture was
then cooled to room temperature in a water bath and ethanol was
added dropwise until gas evolution ceased. The reaction mixture was
diluted with ethyl acetate (300 mL) and washed with aqueous 10%
sodium carbonate (2.times.200 mL). The aqueous phases were combined
and back extracted with ethyl acetate (2.times.100 mL). The organic
phases were combined, dried over MgSO.sub.4, filtered and
concentrated. The resultant oil was purified via column
chromatography (10% EtOAc/Hexanes, 3% TEA) to afford 3.0 g (81%) of
a colorless oil.
3-Dimethylamino-2-(cholest-5-en-3.beta.-oxypent-3-oxa-an-5-oxy)-1-(cis,cis-
-9,12-octadecadienoxy)propane (DEGCLinDMA) (8b)
[0679] This compound was prepared similarly to
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-(cis,cis-9,12-o-
ctadecadienoxy)propane.
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxypent-3-oxa-an-5-oxy)-1-propano-
l (0.73 g, 1.3 mmol) was weighed into a 100 mL round bottomed flask
and co-evaporated with anhydrous toluene. A stir bar was added to
the flask, which was then sealed, flushed with argon and charged
with anhydrous toluene. Sodium hydride (121 mg, 4 equiv.) was added
at once and the mixture was stirred, under argon, for 20 minutes.
Linoleyl mesylate (0.873 g, 2 equiv.) was measured in a PP syringe
and added slowly to the reaction mixture. The flask was fitted with
a reflux condenser and the apparatus was flushed with argon. The
reaction mixture was heated in an oil bath and allowed to stir at
reflux overnight. The reaction mixture was then cooled to room
temperature in a water bath and ethanol was added dropwise until
gas evolution ceased. The reaction mixture was diluted with ethyl
acetate (150 mL) and washed with aqueous 10% sodium carbonate
(2.times.100 mL). The aqueous phases were combined and back
extracted with ethyl acetate (2.times.50 mL). The organic phases
were combined, dried over Na.sub.2SO.sub.4, filtered and
concentrated. The resultant oil was purified via column
chromatography (15% EtOAc/Hexanes, 3% TEA) to afford 0.70 g (67%)
of colorless oil.
Alternative Route for Synthesis of CLinDMA (FIG. 23B)
1-(t-Butyldimethylsilyloxy)-3-dimethylamino-2-propanol (6)
[0680] 3-Dimethylamino-1,2-propanediol (5), (50.1 g, 420.4 mmol)
was weighed into a 2 L round bottomed flask with a stir bar. The
flask was sealed, flushed with argon, charged with
N,N-dimethylformamide (750 mL) and N,N-diisopropylethylamine (111
mL, 630.7 mmol) and cooled to 0.degree. C.
t-Butyldimethylchlorosilane (67.0 g, 1.05 equiv.) was weighed into
a 500 mL round bottomed flask, sealed and then dissolved in
N,N-dimethylformamide (250 mL). The t-butyldimethylchlorosilane
solution was transferred to a pressure equalizing dropping funnel
and added to the stirring reaction mixture slowly over 20 minutes.
The reaction was allowed to come to room temperature while stirring
over 3 hours. The reaction was concentrated in vacuo. Saturated
bicarbonate (1500 mL) was added to the residue and the mixture
transferred to a 4 L separatory funnel. The aqueous phase was
extracted with ethyl acetate (3.times.500 mL). The organic phases
were combined, dried over MgSO.sub.4, filtered, concentrated and
dried under high vacuum to afford 97.81 g (99.7%) of clear,
colorless oil that was used without further purification.
3-Dimethylamino-2-(cholest-5-en-3.beta.-oxybutan-4-oxy)-1-propanol
(7)
[0681] 1-(t-Butyldimethylsilyloxy)-3-Dimethylamino-2-propanol (6)
(25.52 g, 109.3 mmol) was weighed into a two-necked 1 L round
bottomed flask containing a stir bar. The flask was fitted with a
reflux condenser and a ground glass stopper, flushed with argon and
charged with toluene (250 mL). Sodium hydride (10.50 g, 4 equiv.)
was added at once and the mixture was stirred at room temperature
for 20 minutes. Cholest-5-en-3.beta.-oxybutan-4-mesylate (4,
prepared as described above) was dissolved in anhydrous toluene
(100 mL) and added to the reaction mixture at once. An additional
wash of toluene (30 mL) was used to facilitate the complete
transfer of residual mesylate to the reaction mixture. The flask
was subjected to a continuous argon stream and the reaction was
heated to reflux for 8 hrs. The reaction mixture was cooled to
0.degree. C. in a water bath, diluted with ethyl acetate (350 mL)
and ethanol was added dropwise until gas evolution ceased. The
reaction mixture was diluted with more ethyl acetate (350 mL) and
washed with aqueous 10% sodium carbonate (2.times.1 L). The aqueous
phases were combined and back extracted with ethyl acetate
(2.times.500 mL). The organic phases were combined, dried over
MgSO.sub.4, filtered and concentrated to an oil in a 2 L round
bottomed flask.
[0682] The flask was fitted with a stir bar and the residue
dissolved in a mixture of dioxane (300 mL), ethanol (200 mL) and
water (6 mL). Concentrated hydrochloric acid (11.3 mL, 139.2 mmoL)
was added to the solution which was then stirred for 2 hours at
room temperature. 10% Sodium carbonate solution (2 L) was added to
the reaction mixture in a 4 L separatory funnel. The aqueous phase
was extracted with ethyl acetate (3.times.750 mL). The organic
phases were combined and dried over MgSO.sub.4, filtered and
concentrated to an oil. Purification of the oil was performed on a
4.5'' silica gel column pre-equilibrated with 3% TEA in hexanes.
Elution was performed with 1 L of hexanes followed by 3 L of 25%
EtOAc/Hexanes with 3% TEA to afford 28.01 g (50.0%) of a colorless
wax.
Linoleyl mesylate (8)
[0683] Linoleyl alcohol (10.0 g, 37.5 mmol) was weighed into a 500
mL round bottomed flask with a stir bar. The flask was sealed,
flushed with argon, charged with DCM (100 mL) and triethylamine
(7.84 mL, 1.5 equiv.) and cooled to 0.degree. C. Methanesulfonyl
chloride (4.35 mL), 1.5 equiv.) was measured in a PP syringe and
added slowly to the stirring reaction mixture. TLC analysis (7.5%
EtOAc/Hexanes) showed the reaction was complete within 1 hr. The
reaction was diluted with DCM (100 mL) and washed with saturated
bicarbonate solution (2.times.200 mL). The organic phase was dried
over MgSO.sub.4, filtered and concentrated to give 12.53 g (97%) of
colorless oil that was used without further purification.
3-Dimethylamino-2-(cholest-5-en-3.beta.-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-
tadecadienoxy)propane (CLinDMA) (9)
[0684]
3-Dimethylamino-2-(Cholest-5-en-3.beta.-oxybutan-4-oxy)-1-propanol
(7) (2.6 g, 4.6 mmol) was weighed into a 200 mL round bottomed
flask and co-evaporated with anhydrous toluene 2.times.20 mL). A
stir bar was added to the flask, which was then sealed, flushed
with argon and charged with anhydrous toluene (100 mL). Sodium
hydride (0.7 g, 6 equiv) was added at once and the mixture was
stirred, under argon, for 20 minutes. Linoleyl mesylate (4.6 g, 2.3
equiv.) was measured in a PP syringe and added slowly to the
reaction mixture. The flask was fitted with a reflux condenser and
the apparatus was flushed with argon. The reaction mixture was
heated in an oil bath and allowed to stir at reflux overnight. The
reaction mixture was then cooled to room temperature in a water
bath and ethanol was added dropwise until gas evolution ceased. The
reaction mixture was diluted with ethyl acetate (300 mL) and washed
with aqueous 10% sodium carbonate (2.times.200 mL). The aqueous
phases were combined and back extracted with ethyl acetate
(2.times.100 mL). The organic phases were combined, dried over
MgSO.sub.4, filtered and concentrated. The resultant oil was
purified via column chromatography (10% EtOAc/Hexanes, 3% TEA) to
afford 3.0 g (81%) of a colorless oil.
Example 15
Preparation of Aromatic Lipids of the Invention (see FIG. 23C)
Dioleyloxybenzaldehyde, 3a
[0685] 3,4-Dihydroxybenzaldehyde (2.76 g, 20.0 mmol)was weighed
into a 200 mL round bottomed flask with a stir bar. The flask was
charged with diglyme (100 mL), septum sealed and flushed with
argon. Cesium carbonate (19.5 g, 60.0 mmol) was added to the
solution slowly in portions. Oleyl mesylate (15.2 g, 44.0 mmol) was
added via syringe. The reaction mixture was heated to 100.degree.
C. under slight positive pressure of argon. The reaction mixture
was cooled to room temperature and filtered. The solids were washed
with 1,2-dichloroethane. The combined filtrate and washes were
concentrated and then dried under high vacuum at 65.degree. C. to
remove residual diglyme. The resultant yellow oil was purified via
flash chromatography (5% ethyl acetate in hexanes) to afford 11.4 g
(89%) of a yellow oil that turned to yellow wax upon standing at
room temperature.
Dilinoleylbenzaldehyde, 3b
[0686] 3,4-Dihydroxybenzaldehyde (2.76 g, 20.0 mmol)was weighed
into a 200 mL round bottomed flask with a stir bar. The flask was
charged with diglyme (100 mL), septum sealed and flushed with
argon. Cesium carbonate (19.5 g, 60.0 mmol) was added to the
solution slowly in portions. Linoleyl mesylate (15.2 g, 44.0 mmol)
was added via syringe. The reaction mixture was heated to
100.degree. C. under slight positive pressure of argon. The
reaction mixture was cooled to room temperature and filtered. The
solids were washed with 1,2-dichloroethane. The combined filtrate
and washes were concentrated and then dried under high vacuum at
65.degree. C. to remove residual diglyme. The resultant yellow oil
was purified via flash chromatography (5% ethyl acetate in hexanes)
to afford 11.9 g (94%) of a brown oil.
N,N-Dimethyl-3,4-dioleyloxybenzylamine, 4a
[0687] To a solution of triethylamineamine (2.0 mL, 14 mmol) in
ethanol (20 mL) was added dimethylamine hydrochloride (1.63 g, 20
mmol), titanium tetraisopropoxide (5.96 mL, 20 mmol) and
3,4-dioleyloxybenzaldehyde (6.39 g, 10 mmol). The mixture was
allowed to stir under argon for 10 h at room temperature. Sodium
borohydride (0.57 g, 15 mmol) was added to the reaction mixture
which was then allowed to stir at room temperature overnight.
Concentrated aqueous ammonia (4 mL) was added slowly to the
reaction mixture. The reaction mixture was filtered and the solids
washed with dichloromethane. The filtrate was dried over
K.sub.2CO.sub.3, filtered and concentrated. The resultant oil was
purified via flash chromatography (2-10% acetone in
dichloromethane, 0.5% TEA gradient) to afford 5.81 g (87.+-. %) of
a yellow oil.
N,N-Dimethyl-3,4-dilinoleyloxybenzylamine, 4b
[0688] To a solution of triethylamineamine (2.0 mL, 14 mmol) in
ethanol (20 mL) was added dimethylamine hydrochloride (1.63 g, 20
mmol), titanium tetraisopropoxide (5.96 mL, 20 mmol) and
3,4-dilinoleyloxybenzaldehyde (6.35 g, 10 mmol). The mixture was
allowed to stir under argon for 10 h at room temperature. Sodium
borohydride (0.57 g, 15 mmol) was added to the reaction mixture
which was then allowed to stir at room temperature overnight. 6N
Aqueous ammonia (30 mL), was added slowly to the reaction mixture
followed by dichloromethane. The reaction mixture was filtered. The
filtrate was dried over K.sub.2CO.sub.3, filtered and concentrated.
The resultant oil was purified via flash chromatography (2-10%
acetone in dichloromethane, 0.5% TEA gradient) to afford 4.94 g
(74%) of a yellow oil.
Example 16
Preparation of PEG-Conjugates of the Invention (see FIGS. 24A and
24B)
PEG-DMB (FIG. 24A)
1-[8'-(Cholest-5-en-3.beta.-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-.-
omega.-methyl-poly(ethylene glycol) (PEG-cholesterol)
[0689] To a 200-mL round-bottom flask charged with a solution of
2.0 g (0.89 mmol) of
1-[8'-ammino-3',6'-dioxaoctanyl]carbamoyl-.omega.-methyl-poly(ethylene
glycol), 22 mg (0.18 mmol) of 4-dimethylaminopyridine, and 0.93 mL
(5.3 mmol) of diisopropylethylamine in 20 mL of anhydrous THF, was
added with stirring a solution of 1.20 g (2.67 mmol) of cholesterol
chloroformate in 20 mL of anhydrous THF. The resulting reaction
mixture was heated to gentle reflux overnight. After cooled, the
solvents were removed by rotary evaporation, and the resulting
residue was applied onto a silica gel column for purification
(methanol/dichloromethane 5:95 to 10:90). The chromatography
yielded 2.43 g (91%) of white solid product.
3,4-Ditetradecoxylbenzyl-.omega.-methyl-poly(ethylene glycol)ether
(PEG-DMB)
[0690] To a 100-mL round-bottom flask charged with a solution of
2.67 g (5.00 mmol) of ditetradecoxylbenzyl alcohol in 20 mL of
1,4-dioxane, was added 20 mL of 4.0 M HCl solution in 1,4-dioxane.
The flask was then equipped with a refluxing condenser, which was
connected to a sodium bicarbonate solution to absorb any evolved
hydrogen chloride gas. After the reaction mixture was heated to 80
for 6 h, thin layer chromatography (dichloromethane as developing
solvent) indicated the completion of the reaction. The solvent and
the excessive reagent were completely removed under vacuum by
rotary evaporation to afford 2.69 g (97%) of gray solid
3,4-ditetradecoxylbenzyl chloride. This crude material was employed
directly for the next step reaction without further
purification.
[0691] Poly(ethylene glycol) methyl ether (2.00 g, 1.00 mmol) was
dried by co-evaporating with toluene (2.times.20 mL) under vacuum.
The PEG utilized is PEG2000, a polydispersion which can typically
vary from .about.1500 to .about.3000 Da (i.e., where PEG(n) is
about 33 to about 67, or on average .about.45). To a solution of
the dried poly(ethylene glycol) in 30 mL of anhydrous toluene, was
added with stirring 0.17 g (7.2 mmol) of sodium hydride in
portions. Gas evolvement took place instantly. The resulting
mixture continued to be stirred at 60 for 2 h to ensure the
complete formation of oxide. A solution of 0.668 g (1.20 mmol)
3,4-ditetradecoxylbenzyl chloride in 10 mL of anhydrous toluene was
then introduced dropwise to the above mixture. The reaction mixture
was allowed to stir at 80 overnight. After cooled, the reaction was
quenched by the addition of 10 mL of saturated ammonium chloride
solution. The resulting mixture was then taken into 300 mL of
dichloromethane, washed with saturated ammonium chloride
(3.times.100 mL), dried over anhydrous sodium sulfate, and
evaporated to dryness. The residue was purified by flash
chromatography (methanol/dichloromethane 2:98 to 5:95) to furnish
1.24 g (49%) of gray solid of the desired product.
PEG-DMG (FIG. 24B)
[0692] 1,2-dimyristoyl-sn-glycerol (DMG-OH) (1) (10.0 g) and
1,1'-Carbonyldiimidazole (CDI) (2) (3.32 g, 1.05 eq) were added to
a 250 mL dry round bottom flask equipped with magnetic stir bar and
rubber septa under argon. The flask was charged with 50 mL
anhydrous THF and the resulting mixture stirred for 6 hours at room
temperature. The stir bar was removed and the reaction mixture
transferred to a 1 L separatory funnel with 350 mL ethyl acetate.
The reaction mixture was washed with 200 mL deionized water. The
aqueous phase was removed and the wash repeated 2.times., the
organic phase was collected and dried over 10 g magnesium sulphate
with stirring. Filtration over sintered glass followed by
evaporation in vacuo provided
1,2-Dimyristoyl-3-propanoxy-carboximidazole (DMG-CDI) (3); 11.68 g,
99%. To a mixture of Methoxy-PEG-NH2 2K (PEG-amine) (4) (2.36 g);
1,2-Dimyristoyl-3-propanoxy-carboximidazole (DMG-CDI) (3) (1.91 g,
3.0 eq); and 4-(N,N-Dimethylamino)pyridine (DMAP) (0.025 g, 0.2 eq)
in a 200 mL round bottomed flask equipped with stir bar and rubber
septum under argon was added THF (20 mL) and Diisopropylethylamine
(DiPEA) (1.10 mL, 6.0 eq). The PEG utilized is PEG2000, a
polydispersion which can typically vary from .about.1500 to
.about.3000 Da (i.e., where PEG(n) is about 33 to about 67, or on
average .about.45). The solution was brought to reflux and stirred
for 17 hours after which the reaction mixture was cooled to room
temperature, the stir bar removed, and the reaction concentrated in
vacuo to provide crude
1-[8'-(1,2-Dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl]carbam-
oyl-.omega.-methyl-poly(ethylene glycol) (PEG-DMG) (5), 4.31 g.
Example 17
Preparation of Nanoparticle Encapsulated siNA Formulations
General LNP Preparation
[0693] siNA nanoparticle solutions were prepared by dissolving
siNAs in 25 mM citrate buffer (pH 4.0) at a concentration of 0.9
mg/mL. Lipid solutions were prepared by dissolving a mixture of
cationic lipid (e.g., CLinDMA or DOBMA, see structures and ratios
for Formulations in Table IV), DSPC, Cholesterol, and PEG-DMG
(ratios shown in Table IV) in absolute ethanol at a concentration
of about 15 mg/mL. The nitrogen to phosphate ratio was approximate
to 3:1.
[0694] Equal volume of siNA and lipid solutions was delivered with
two FPLC pumps at the same flow rates to a mixing T connector. A
back pressure valve was used to adjust to the desired particle
size. The resulting milky mixture was collected in a sterile glass
bottle. This mixture was then diluted slowly with an equal volume
of citrate buffer, and filtered through an ion-exchange membrane to
remove any free siNA in the mixture. Ultra filtration against
citrate buffer (pH 4.0) was employed to remove ethanol (test stick
from ALCO screen), and against PBS (pH 7.4) to exchange buffer. The
final LNP was obtained by concentrating to a desired volume and
sterile filtered through a 0.2 .mu.m filter. The obtained LNPs were
characterized in term of particle size, Zeta potential, alcohol
content, total lipid content, nucleic acid encapsulated, and total
nucleic acid concentration
LNP Manufacture Process
[0695] In a non-limiting example, a LNP-086 siNA formulation is
prepared in bulk as follows. A process flow diagram for the process
is shown in Table VI which can be adapted for siNA coctails (2 siNA
duplexes are shown) or for a single siNA duplex. The process
consists of (1) preparing a lipid solution; (2) preparing a siNA
solution; (3) mixing/particle formation; (4) Incubation; (5)
Dilution; (6) Ultrafiltraion and Concentration.
1. Preparation of Lipid Solution
[0696] Summary: To a 3-necked round bottom flask fitted with a
condenser was added a mixture of CLinDMA, DSPC, Cholesterol,
PEG-DMG, and Linoeyl alcohol. Ethanol was then added. The
suspension was stirred with a stir bar under Argon, and was heated
at 30.degree. C. using a heating mantle controlled with a process
controller. After the suspension became clear, the solution was
allowed to cool to room temperature.
Detailed Procedure for Formulating 8 L Batch of LNP
[0697] 1. Depyrogenate a 3-necked 2 L round bottom flask, a
condenser, measuring cylinders, and two 10 L conical glass vessels.
[0698] 2. Warm the lipids to room temperature. Tare the weight of
the round bottom flask. Transfer the CLinDMA (50.44 g) with a
pipette using a pipette aid into the 3-necked round bottom flask.
[0699] 3. Weigh DSPC (43.32 g), Cholesterol (5.32 g) and PEG-DMG
(6.96 g) with a weighing paper sequentially into the round bottom
flask. [0700] 4. Linoleyl alcohol (2.64 g) was weighed in a
separate glass vial (depyrogenated). Tare the vial first, and then
transfer the compound with a pipette into the vial. [0701] 5. Take
the total weight of the round bottom flask with the lipids in,
subtract the tare weight. The error was usually much less than
.+-.1.0%. [0702] 6. Transfer one-eighth of the ethanol (1 L) needed
for the lipid solution into the round bottom flask. [0703] 7. The
round bottom flask placed in a heating mantle was connected to a
J-CHEM process controller. The lipid suspension was stirred under
Argon with a stir bar and a condenser on top. A thermocouple probe
was put into the suspension through one neck of the round bottom
flask with a sealed adapter. [0704] 8. The suspension was heated at
30.degree. C. until it became clear. The solution was allowed to
cool to room temperature and transferred to a conical glass vessel
and sealed with a cap. 2. Preparation of siNA Solution
[0705] Summary: The siNA solution can comprise a single siNA duplex
or can alternately comprise a cocktail of two or more siNA
duplexes. In the case of a single siNA duplex, the siNA is
dissolved in 25 mM citrate buffer (pH 4.0, 100 mM of NaCl) to give
a final concentration of 0.9 mg/mL. In the case of a cocktail of
two siNA molecules, the siNA solutions are prepared by dissolving
each siNA molecule in 50% of the total expected volume of a 25 mM
citrate buffer (pH 4.0, 100 mM of NaCl) to give a final
concentration of 0.9 mg/mL. This procedure is repeated for the
other sNA molecule. The two 0.9 mg/mL siNA solutions are combined
to give a 0.9 mg/mL solution at the total volume containing two
siNA molecules.
Detailed Procedure for Formulating 8 L Batch of LNP with siNA
Cocktail [0706] 1. Weigh 3.6 g times the water correction factor
(Approximately 1.2) of siNA-1 powder into a sterile container such
as the Corning storage bottle. [0707] 2. Transfer the siNA to a
depyrogenated 5 L glass vessel. Rinse the weighing container
3.times. with of citrate buffer (25 mM, pH 4.0, and 100 mM NaCl)
placing the rinses into the 5 L vessel, QS with citrate buffer to 4
L. [0708] 3. Determine the concentration of the siNA solution with
UV spectrometer. Generally, take 20 .mu.L from the solution, dilute
50 times to 1000 .mu.L, and record the UV reading at A260 nm after
blanking with citrate buffer. Make a parallel sample and measure.
If the readings for the two samples are consistent, take an average
and calculate the concentration based on the extinction
coefficients of the siNAs. If the final concentration is out of the
range of 0.90.+-.0.01 mg/mL, adjust the concentration by adding
more siNA powder, or adding more citrate buffer. [0709] 4. Repeat
for siNA-2. [0710] 5. In a 10 l depyrogenated 10 L glass vessel
transfer 4 L of each 0.9 mg/mL siNA solution
Sterile Filtration.
[0711] The process describes the procedure to sterile filter the
Lipid/Ethanol solution. The purpose is to provide a sterile
starting material for the encapsulation process. The filtration
process was run at an 80 mL scale with a membrane area of 20
cm.sup.2. The flow rate is 280 mL/min. This process is scaleable by
increasing the tubing diameter and the filtration area.
[0712] 1. Materials [0713] a. Nalgene 50 Silicone Tubing PN
8060-0040 Autoclaved [0714] b. Master Flex Peristaltic Pump Model
7520-40 [0715] i. Master flex Pump Head Model 7518-00 [0716] c.
Pall Acropak 20 0.8/0.2 .mu.m sterile filter. PN 12203 [0717] d.
Depyrogenated 10 L glass vessel [0718] e. Autoclaved lid for glass
vessel.
[0719] 2. Procedure. [0720] a. Place tubing into pump head. Set
pump to 50% total pump speed and measure flow for 1 minute with a
graduated cylinder [0721] b. Adjust pump setting and measure flow
to 280 mL/min. [0722] c. Set up Tubing with filter attach securely
with a clamp. [0723] d. Set up pump and place tubing into pump
head. [0724] e. Place the feed end of the tubing into the material
to be filtered. [0725] f. Place the filtrate side of filter with
filling bell into depyrogenated glass vessel. [0726] g. Pump
material through filter until all material is filtered.
AKTA Pump Setup
[0727] 1. Materials [0728] a. AKTA P900 Pump [0729] b. Teflon
tubing 2 mm ID.times.3 mm OD 2 each.times.20.5 cm Upchurch PN 1677
[0730] c. Teflon tubing 1 mm ID.times.3 mm OD 6.5 cm Upchurch PN
1675 [0731] d. Peek Tee 1 mm ID 1 each Upchurch PN P-714 [0732] e.
1/4-28 F to 10-32M 2 each Upchurch PN P-652 [0733] f. ETFE Ferrule
for 3.0 mm OD tubing 6 each Upchurch PN P-343x [0734] g. Flangless
Nut 6 each Upchurch PN P-345x [0735] h. ETFE cap for 1/4-28 flat
bottom fitting 1 each Upchurch PN P-755 [0736] i. Argon Compressed
gas [0737] j. Regulator 0-60 psi [0738] k. Teflon tubing [0739] l.
Peek Y fitting [0740] m. Depyrogenated glassware conical
base.2/pump [0741] n. Autoclaved lids. [0742] o. Pressure lids
[0743] 2. Pump Setup [0744] a. Turn pump on [0745] b. Allow pump to
perform self test [0746] c. Make certain that there are no caps or
pressure regulators attached to tubing (This will cause the pumps
to over pressure.) [0747] d. Press "OK" to synchronize pumps [0748]
e. Turn knob 4 clicks clockwise to "Setup"--press "OK" [0749] f.
Turn knob 5 clicks clockwise to "Setup Gradient Mode"--press "OK"
[0750] g. Turn knob 1 click clockwise to "D"--press "OK" [0751] h.
Press "Esc" twice
[0752] 3. Pump Sanitization. [0753] a. Place 1000 mL of 1 N NaOH
into a 1 L glass vessel [0754] b. Attach to pump with a pressure
lid [0755] c. Place 1000 mL of 70% Ethanol into a 1 L glass vessel
[0756] d. Attach to pump with a pressure lid. [0757] e. Place a
2000 mL glass vessel below pump outlet. [0758] f. Turn knob 1 click
clockwise to "Set Flow Rate"--press "OK" [0759] g. Turn knob
clockwise to increase Flow Rate to 40 mL/min; counter clockwise to
decrease; press "OK" when desired Flow Rate is set. [0760] h. Set
time for 40 minute. [0761] i. Turn on argon gas at 10 psi. [0762]
j. Turn knob 2 clicks counter clockwise to "Run"--press "OK", and
start timer. [0763] k. Turn knob 1 click counter clockwise to "End
Hold Pause" [0764] l. When timer sounds Press "OK" on pump [0765]
m. Turn off gas [0766] n. Store pump in sanitizing solutions until
ready for use (overnight?)
[0767] 4. Pump Flow Check [0768] a. Place 200 mL of Ethanol into a
depyrogenated 500 mL glass bottle. [0769] b. Attach to pump with a
pressure cap. [0770] c. Place 200 mL of Sterile Citrate buffer into
a 500 mL depyrogenated glass bottle. [0771] d. Attach to pump with
a pressure cap. [0772] e. Place a 100 mL graduated cylinder below
pump outlet. [0773] f. Turn knob 1 click clockwise to "Set Flow
Rate"--press "OK" [0774] g. Turn knob clockwise to increase Flow
Rate to 40 mL/min; counter clockwise to decrease; press "OK" when
desired Flow Rate is set. [0775] h. Set time for 1 minute. [0776]
i. Turn on argon gas at 10 psi. [0777] j. Turn knob 2 clicks
counter clockwise to "Run"--press "OK", and start timer. [0778] k.
Turn knob 1 click counter clockwise to "End Hold Pause" [0779] l.
When timer sounds Press "OK" on pump [0780] m. Turn off gas [0781]
n. Verify that 40 mL of the ethanol/citrate solution was
delivered.
3. Particle Formation--Mixing Step
[0781] [0782] o. Attach the sterile Lipid/Ethanol solution to the
AKTA pump. [0783] p. Attach the sterile siNA or siNA
cocktail/Citrate buffer solution to the AKTA pump. [0784] q. Attach
depyrogenated received vessel (2.times. batch size) with lid [0785]
r. Set time for calculated mixing time. [0786] s. Turn on Argon gas
and maintain pressure between 5 to10 psi. [0787] t. Turn knob 2
clicks counter clockwise to "Run"--press "OK", and start timer.
[0788] u. Turn knob 1 click counter clockwise to "End Hold Pause"
[0789] v. When timer sounds Press "OK" on pump [0790] w. Turn off
gas
4. Incubation
[0791] The solution is held after mixing for a 22 .+-.2 hour
incubation. The incubation is at room temperature (20-25.degree.
C.) and the in-process solution is protected from light.
5. Dilution.
[0792] The lipid siNA solution is diluted with an equal volume of
Citrate buffer. The solution is diluted with a dual head
peristaltic pump, set up with equal lengths of tubing and a Tee
connection. The flow rate is 360 mL/minute.
[0793] 1. Materials [0794] h. Nalgene 50 Silicone Tubing PN
8060-0040 Autoclaved [0795] i. Tee 1/4'' ID [0796] j. Master Flex
Peristaltic Pump Model 7520-40 [0797] i. Master flex Pump Head
Model 7518-00 [0798] ii. Master flex Pump Head Model 7518-00 [0799]
k. Depyrogenated 2.times.20 L glass vessel [0800] l. Autoclaved
lids for glass vessels.
[0801] 2. Procedure. [0802] a. Attach two equal lengths of tubing
to the Tee connector. The tubing should be approximately 1 meter in
length. Attach a third piece of tubing approximately 50 cm to the
outlet end of the Tee connector. [0803] b. Place the tubing
apparatus into the dual pump heads. [0804] c. Place one feed end of
the tubing apparatus into an Ethanol solution. Place the other feed
end into an equal volume of Citrate buffer. [0805] d. Set the pump
speed control 50%. Set a time for 1 minute. [0806] e. Place the
outlet end of the tubing apparatus into a 500 mL graduated
cylinder. [0807] f. Turn on the pump and start the timer. [0808] g.
When the timer sounds stop the pump and determine the delivered
volume. [0809] h. Adjust the pump flow rate to 360 mL/minute.
[0810] i. Drain the tubing when the flow rate is set. [0811] j.
Place one feed end of the tubing apparatus into the Lipid/siNA
solution. Place the other feed end into an equal volume of Citrate
buffer (16 L). [0812] k. Place the outlet end of the tubing
apparatus into the first of 2.times.20 L depyrogenated glass
vessels. [0813] l. Set a timer for 90 minutes and start the pump.
Visually monitor the dilution progress to ensure that the flow
rates are equal. [0814] m. When the receiver vessel is at 16 liters
change to the next vessel and collect 16 L. [0815] n. Stop the pump
when all the material has been transferred.
6. Ultrafiltration and Concentration
[0816] Summary: The ultrafiltration process is a timed process and
the flow rates must be monitored carefully. The membrane area has
been determined based on the volume of the batch. This is a two
step process; the first is a concentration step taking the diluted
material from 32 liters to 3600 mLs and a concentration of
approximately 2 mg/mL. The concentration step is 4 hours .+-.15
minutes. The second step is a diafiltration step exchanging the
ethanol citrate buffer to Phosphate buffered saline. The
diafiltration step is 3 hours and again the flow rates must be
carefully monitored. During this step the ethanol concentration is
monitored by head space GC. After 3 hours (20 diafiltration
volumes) a second concentration is undertaken to concentrate the
solution to approximately 6 mg/mL or a volume of 1.2 liters. This
material is collected into a depyrogenated glass vessel. The system
is rinsed with 400 mL of PBS at high flow rate and the permeate
line closed. This material is collected and added to the first
collection. The expected concentration at this point is 4.5 mg/mL.
The concentration and volume are determined.
[0817] 1. Materials [0818] x. Quatroflow pump [0819] y. Flexstand
system with autoclaved 5 L reservoir. [0820] z. Ultrafiltration
membrane GE PN UFP-100-C-35A [0821] aa. PBS 0.05.mu.m filtered 100
L [0822] bb. 0.5 N Sodium Hydroxide. [0823] cc. WFI [0824] dd.
Nalgene 50 Silicone Tubing PN 8060-0040 Autoclaved [0825] ee.
Master Flex Peristaltic Pump Model 7520-40 [0826] i. Master flex
Pump Head Model 7518-00 [0827] ff. Permeate collection vessels 100
L capacity [0828] gg. Graduated cylinders depyrogenated 2 L, 11,
500 mL.
[0829] 2. Procedure
[0830] a. System Preparation. [0831] i. Install the membrane in the
Flexstand holder, using the appropriate size sanitary fittings for
the membrane. Attach the Flexstand to the quatroflow pump. Attach
tubing to the retentate and permeate connections and place these in
a suitable waste container. [0832] ii. Determine the system hold up
volume. [0833] 1. Place 1 liter of WFI in the reservoir. [0834] 2.
Clamp the permeate line. [0835] 3. Start the Quatroflow pump and
recirculate until no bubbles are present in the retentate line.
Stop pump [0836] 4. Mark the reservoir and record the reading for 1
liter. [0837] 5. Add 200 mL of WFI to the reservoir and mark the
1200 mL level. [0838] iii. Add 3 liters of 0.5 N sodium hydroxide
to the reservoir and flush through the retentate to waste. Add 3 L
of 0.5 N sodium hydroxide to the reservoir recirculate the
retentate line and flush through the permeate to waste. Add a third
3 L of 0.5 N sodium hydroxide to the reservoir and recirculate
through the permeate line to the reservoir for 30 minutes. Store
the system in 0.5 N sodium hydroxide overnight prior to use. [0839]
iv. Flush the sodium hydroxide to waste. [0840] v. Add 3 L WFI to
the reservoir and flush the retentate to waste until the pH is
neutral, replace the WFI as necessary. Return the retentate line to
the reservoir. [0841] vi. Add 3 Liters of WFI and flush the
permeate line to waste until the pH is neutral, replacing the WFI
as necessary. Drain system. [0842] vii. Add 3 Liters of Citrate
buffer to the reservoir. Flush through the permeate line until pH
is <5. Add citrate buffer as necessary. [0843] viii. Drain
system.
[0844] b. LNP Concentration [0845] i. Place a suitable length on
tubing into the peristaltic pump head. [0846] ii. Place the feed
end into the diluted LNP solution; place the other end into the
reservoir. [0847] iii. Pump the diluted LNP solution into the
reservoir to the 4 liter mark. [0848] iv. Place the permeate line
into a clean waste container. [0849] v. Start the quatroflow pump
and adjust the pump speed so the permeate flow rate is 300 mL/min.
[0850] vi. Adjust the peristaltic pump to 300 ml/min so the liquid
level is constant at 4 L in the reservoir. [0851] vii. When all the
diluted LNP solution has been transferred to the reservoir stop the
peristaltic pump. [0852] viii. Concentrate the diluted LNP solution
to 3600 mL in 240 minutes by adjusting the pump speed as necessary.
[0853] ix. Monitor the permeate flow rate, pump setting and feed
and retentate pressures.
[0854] c. LNP Diafiltration [0855] i. Place the feed tubing of the
peristaltic pump into a container containing 72 L of PBS (0.05
.mu.m filtered). [0856] ii. Start the peristaltic pump and adjust
the flow rate to maintain a constant volume of 3600 mL in the
reservoir. [0857] iii. Increase the Quatroflow pump flow rate to
400 mL/min. [0858] iv. Monitor the permeate flow rate, pump setting
and feed and retentate pressures. [0859] v. Monitor the ethanol
concentration by GC [0860] vi. The LNP solution is diafiltred with
PBS (20 volumes) for 180 minutes. [0861] vii. Stop the peristaltic
pump. Remove tubing from reservoir.
[0862] d. Final concentration [0863] i. Concentrate the LNP
solution to the 1.2 Liter mark. [0864] ii. Collect the LNP solution
into a depyrogenated 2 L graduated cylinder. [0865] iii. Add 400 mL
of PBS to the reservoir. [0866] iv. Start the pump and recirculate
for 2 minutes. [0867] v. Collect the rinse and add to the collected
LNP solution in the graduated cylinder. [0868] vi. Record the
volume of the LNP solution. [0869] vii. Transfer to a 2 L
depyrogenated glass vessel. [0870] viii. Label and refrigerate.
[0871] e. Clean System [0872] i. Add 1 L WFI to the reservoir
[0873] ii. Recirculate for 5 minutes with permeate closed. [0874]
iii. Drain system [0875] iv. Add 2 L 0.5 N sodium hydroxide to the
reservoir [0876] v. Recirculate for 5 minutes. [0877] vi. Drain
system [0878] vii. Add 2 L of 0.5 N sodium hydroxide to the
reservoir. [0879] viii. Recirculate for 5 minutes and stop pump.
[0880] ix. Neutralize system with WFI. [0881] x. Drain system and
discard membrane.
[0882] The obtained LNPs were characterized in term of particle
size, Zeta potential, alcohol content, total lipid content, nucleic
acid encapsulated, and total nucleic acid concentration.
[0883] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. All references cited in this
disclosure are incorporated by reference to the same extent as if
each reference had been incorporated by reference in its entirety
individually.
[0884] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods and compositions described herein as presently
representative of preferred embodiments are exemplary and are not
intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art,
which are encompassed within the spirit of the invention, are
defined by the scope of the claims.
[0885] It will be readily apparent to one skilled in the art that
varying substitutions and modifications can be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. Thus, such additional embodiments are
within the scope of the present invention and the following claims.
The present invention teaches one skilled in the art to test
various combinations and/or substitutions of chemical modifications
described herein toward generating nucleic acid constructs with
improved activity for mediating RNAi activity. Such improved
activity can comprise improved stability, improved bioavailability,
and/or improved activation of cellular responses mediating RNAi.
Therefore, the specific embodiments described herein are not
limiting and one skilled in the art can readily appreciate that
specific combinations of the modifications described herein can be
tested without undue experimentation toward identifying siNA
molecules with improved RNAi activity.
[0886] The invention illustratively described herein suitably can
be practiced in the absence of any element or elements, limitation
or limitations that are not specifically disclosed herein. The
terms and expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments, optional features,
modification and variation of the concepts herein disclosed may be
resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention as defined by the description and the appended
claims.
[0887] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other
group.
TABLE-US-00002 TABLE I Non-limiting examples of Stabilization
Chemistries for chemically modified siNA constructs Chemistry
pyrimidine Purine cap p = S Strand "Stab 00" Ribo Ribo TT at
3'-ends S/AS "Stab 1" Ribo Ribo -- 5 at 5'-end S/AS 1 at 3'-end
"Stab 2" Ribo Ribo -- All linkages Usually AS "Stab 3" 2'-fluoro
Ribo -- 4 at 5'-end Usually S 4 at 3'-end "Stab 4" 2'-fluoro Ribo
5' and 3'-ends -- Usually S "Stab 5" 2'-fluoro Ribo -- 1 at 3'-end
Usually AS "Stab 6" 2'-O-Methyl Ribo 5' and 3'-ends -- Usually S
"Stab 7" 2'-fluoro 2'-deoxy 5' and 3'-ends -- Usually S "Stab 8"
2'-fluoro 2'-O-Methyl -- 1 at 3'-end S/AS "Stab 9" Ribo Ribo 5' and
3'-ends -- Usually S "Stab 10" Ribo Ribo -- 1 at 3'-end Usually AS
"Stab 11" 2'-fluoro 2'-deoxy -- 1 at 3'-end Usually AS "Stab 12"
2'-fluoro LNA 5' and 3'-ends Usually S "Stab 13" 2'-fluoro LNA 1 at
3'-end Usually AS "Stab 14" 2'-fluoro 2'-deoxy 2 at 5'-end Usually
AS 1 at 3'-end "Stab 15" 2'-deoxy 2'-deoxy 2 at 5'-end Usually AS 1
at 3'-end "Stab 16" Ribo 2'-O-Methyl 5' and 3'-ends Usually S "Stab
17" 2'-O-Methyl 2'-O-Methyl 5' and 3'-ends Usually S "Stab 18"
2'-fluoro 2'-O-Methyl 5' and 3'-ends Usually S "Stab 19" 2'-fluoro
2'-O-Methyl 3'-end S/AS "Stab 20" 2'-fluoro 2'-deoxy 3'-end Usually
AS "Stab 21" 2'-fluoro Ribo 3'-end Usually AS "Stab 22" Ribo Ribo
3'-end Usually AS "Stab 23" 2'-fluoro* 2'-deoxy* 5' and 3'-ends
Usually S "Stab 24" 2'-fluoro* 2'-O-Methyl* -- 1 at 3'-end S/AS
"Stab 25" 2'-fluoro* 2'-O-Methyl* -- 1 at 3'-end S/AS "Stab 26"
2'-fluoro* 2'-O-Methyl* -- S/AS "Stab 27" 2'-fluoro* 2'-O-Methyl*
3'-end S/AS "Stab 28" 2'-fluoro* 2'-O-Methyl* 3'-end S/AS "Stab 29"
2'-fluoro* 2'-O-Methyl* 1 at 3'-end S/AS "Stab 30" 2'-fluoro*
2'-O-Methyl* S/AS "Stab 31" 2'-fluoro* 2'-O-Methyl* 3'-end S/AS
"Stab 32" 2'-fluoro 2'-O-Methyl S/AS "Stab 33" 2'-fluoro 2'-deoxy*
5' and 3'-ends -- Usually S "Stab 34" 2'-fluoro 2'-O-Methyl* 5' and
3'-ends Usually S "Stab 35" 2'-fluoro** 2'-O-Methyl** Usually AS
"Stab 36" 2'-fluoro** 2'-O-Methyl** Usually AS "Stab 3F" 2'-OCF3
Ribo -- 4 at 5'-end Usually S 4 at 3'-end "Stab 4F" 2'-OCF3 Ribo 5'
and 3'-ends -- Usually S "Stab 5F" 2'-OCF3 Ribo -- 1 at 3'-end
Usually AS "Stab 7F" 2'-OCF3 2'-deoxy 5' and 3'-ends -- Usually S
"Stab 8F" 2'-OCF3 2'-O-Methyl -- 1 at 3'-end S/AS "Stab 11F"
2'-OCF3 2'-deoxy -- 1 at 3'-end Usually AS "Stab 12F" 2'-OCF3 LNA
5' and 3'-ends Usually S "Stab 13F" 2'-OCF3 LNA 1 at 3'-end Usually
AS "Stab 14F" 2'-OCF3 2'-deoxy 2 at 5'-end Usually AS 1 at 3'-end
"Stab 15F" 2'-OCF3 2'-deoxy 2 at 5'-end Usually AS 1 at 3'-end
"Stab 18F" 2'-OCF3 2'-O-Methyl 5' and 3'-ends Usually S "Stab 19F"
2'-OCF3 2'-O-Methyl 3'-end S/AS "Stab 20F" 2'-OCF3 2'-deoxy 3'-end
Usually AS "Stab 21F" 2'-OCF3 Ribo 3'-end Usually AS "Stab 23F"
2'-OCF3* 2'-deoxy* 5' and 3'-ends Usually S "Stab 24F" 2'-OCF3*
2'-O-Methyl* -- 1 at 3'-end S/AS "Stab 25F" 2'-OCF3* 2'-O-Methyl*
-- 1 at 3'-end S/AS "Stab 26F" 2'-OCF3* 2'-O-Methyl* -- S/AS "Stab
27F" 2'-OCF3* 2'-O-Methyl* 3'-end S/AS "Stab 28F" 2'-OCF3*
2'-O-Methyl* 3'-end S/AS "Stab 29F" 2'-OCF3* 2'-O-Methyl* 1 at
3'-end S/AS "Stab 30F" 2'-OCF3* 2'-O-Methyl* S/AS "Stab 31F"
2'-OCF3* 2'-O-Methyl* 3'-end S/AS "Stab 32F" 2'-OCF3 2'-O-Methyl
S/AS "Stab 33F" 2'-OCF3 2'-deoxy* 5' and 3'-ends -- Usually S "Stab
34F" 2'-OCF3 2'-O-Methyl* 5' and 3'-ends Usually S "Stab 35F"
2'-OCF3*.dagger. 2'-O-Methyl*.dagger. Usually AS "Stab 36F"
2'-OCF3*.dagger. 2'-O-Methyl*.dagger. Usually AS CAP = any terminal
cap, see for example FIG. 10. All Stab 00-34 chemistries can
comprise 3'-terminal thymidine (TT) residues All Stab 00-34
chemistries typically comprise about 21 nucleotides, but can vary
as described herein. All Stab 00-36 chemistries can also include a
single ribonucleotide in the sense or passenger strand at the
11.sup.th base paired position of the double stranded nucleic acid
duplex as determined from the 5'-end of the antisense or guide
strand (see FIG. 6C) S = sense strand AS = antisense strand *Stab
23 has a single ribonucleotide adjacent to 3'-CAP *Stab 24 and Stab
28 have a single ribonucleotide at 5'-terminus *Stab 25, Stab 26,
Stab 27, Stab 35 and Stab 36 have three ribonucleotides at
5'-terminus *Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any
purine at first three nucleotide positions from 5'-terminus are
ribonucleotides p = phosphorothioate linkage .dagger.Stab 35 has
2'-O-methyl U at 3'-overhangs and three ribonucleotides at
5'-terminus .dagger.Stab 36 has 2'-O-methyl overhangs that are
complementary to the target sequence (naturually occurring
overhangs) and three ribonucleotides at 5'-terminus
TABLE-US-00003 TABLE II A. 2.5 .mu.mol Synthesis Cycle ABI 394
Instrument Reagent Equivalents Amount Wait Time* DNA Wait Time*
2'-O-methyl Wait Time*RNA Phosphoramidites 6.5 163 .mu.L 45 sec 2.5
min 7.5 min S-Ethyl Tetrazole 23.8 238 .mu.L 45 sec 2.5 min 7.5 min
Acetic Anhydride 100 233 .mu.L 5 sec 5 sec 5 sec N-Methyl 186 233
.mu.L 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21
sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 .mu.L
100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2
.mu.mol Synthesis Cycle ABI 394 Instrument Reagent Equivalents
Amount Wait Time* DNA Wait Time* 2'-O-methyl Wait Time*RNA
Phosphoramidites 15 31 .mu.L 45 sec 233 sec 465 sec S-Ethyl
Tetrazole 38.7 31 .mu.L 45 sec 233 min 465 sec Acetic Anhydride 655
124 .mu.L 5 sec 5 sec 5 sec N-Methyl 1245 124 .mu.L 5 sec 5 sec 5
sec Imidazole TCA 700 732 .mu.L 10 sec 10 sec 10 sec Iodine 20.6
244 .mu.L 15 sec 15 sec 15 sec Beaucage 7.7 232 .mu.L 100 sec 300
sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 .mu.mol
Synthesis Cycle 96 well Instrument Equivalents: DNA/ Amount:
DNA/2'-O- Wait Time* 2'-O- Reagent 2'-O-methyl/Ribo methyl/Ribo
Wait Time* DNA methyl Wait Time* Ribo Phosphoramidites 22/33/66
40/60/120 .mu.L 60 sec 180 sec 360 sec S-Ethyl Tetrazole 70/105/210
40/60/120 .mu.L 60 sec 180 min 360 sec Acetic Anhydride 265/265/265
50/50/50 .mu.L 10 sec 10 sec 10 sec N-Methyl 502/502/502 50/50/50
.mu.L 10 sec 10 sec 10 sec Imidazole TCA 238/475/475 250/500/500
.mu.L 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 .mu.L 30 sec
30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec
Acetonitrile NA 1150/1150/1150 .mu.L NA NA NA Wait time does not
include contact time during delivery. Tandem synthesis utilizes
double coupling of linker molecule
TABLE-US-00004 TABLE III Structure NAME Abbrev. ##STR00060##
Cholesterol Chol ##STR00061## Cholest-5-en-3.beta.-tosylate
Chol-OTs ##STR00062## Cholest-5-en-3.beta.-oxybutan-4-ol
Chol-OBu-OH ##STR00063## Cholest-5-en-3.beta.-oxypent-3-oxa-an-5-ol
Chol-DEG-OH ##STR00064## Cholest-5-en-3.beta.-oxybutan-4-mesylate
##STR00065## Cholest-5-en-3.beta.-oxypent-3-oxa-an-5- mesylate
##STR00066## 3-Dimethylamino-1,2-propanediol ##STR00067##
1-(4,4'-Dimethoxytrityloxy)-3- Dimethylamino-2-propanol
##STR00068## 3-Dimethylamino-2-(Cholest-5-en-3.beta.-
oxybutan-4-oxy)-1-propanol ##STR00069##
3-Dimethylamino-2-(Cholest-5-en-3.beta.-
oxypent-3-oxa-an-5-oxy)-1-propanol ##STR00070##
cis,cis-9,12-octadecadiene-1-ol (linoleyl alcohol) Lin-OH
##STR00071## cis,cis-9,12-octadecadiene-1-mesylate (linoleyl
mesylate) Lin-OMs ##STR00072##
3-Dimethylamino-2-(Cholest-5-en-3.beta.-
oxybutan-4-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane CLinDMA
##STR00073## 3-Dimethylamino-2-(Cholest-5-en-3.beta.-
oxypent-3-oxa-an-5-oxy)-1-(cis,cis- 9,12-octadecadienoxy)propane
DEG-CLinDMA
TABLE-US-00005 TABLE IV Lipid Nanoparticle (LNP) Formulations
Formu- lation # Composition Molar Ratio L051
CLinDMA/DSPC/Chol/2KPEG-DMG 48/40/10/2 L053
DMOBA/DSPC/Chol/2KPEG-DMG 30/20/48/2 L054 DMOBA/DSPC/Chol/2KPEG-DMG
50/20/28/2 L065 DEG-CLinDMA/DSPC/Chol/2KPEG-DMG 48/40/10/2 L069
CLinDMA/DSPC/Cholesterol/PEG- 48/40/10/2 Cholesterol L073 pCLinDMA
or CLin DMA/DMOBA/DSPC/ 25/25/20/28/2 Chol/2KPEG-DMG L077
eCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 Chol L080
eCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 DMG L082
pCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 DMG L083
pCLinDMA/DSPC/Cholesterol/2KPEG- 48/40/10/2 Chol L086
CLinDMA/DSPC/Cholesterol/2KPEG- 43/38/10/2/7 DMG/Linoleyl alcohol
L061 DMLBA/Cholesterol/2KPEG-DMG 52/45/3 L060
DMOBA/Cholesterol/2KPEG-DMG N/P ratio 52/45/3 of 5 L097
DMLBA/DSPC/Cholesterol/2KPEG-DMG 50/20/28 L098
DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 3 L099
DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 4 L100
DMOBA/DOBA/3% PEG-DMG, N/P ratio of 52/45/3 3 L101
DMOBA/Cholesterol/2KPEG-Cholesterol 52/45/3 L102
DMOBA/Cholesterol/2KPEG-Cholesterol, 52/45/3 N/P ratio of 5 L103
DMLBA/Cholesterol/2KPEG-Cholesterol 52/45/3 L104
CLinDMA/DSPC/Cholesterol/2KPEG- 43/38/10/2/7 cholesterol/Linoleyl
alcohol L105 DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 52/45/3 of 2
L106 DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 67/30/3 of 3 L107
DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 52/45/3 of 1.5 L108
DMOBA/Cholesterol/2KPEG-Chol, N/P ratio 67/30/3 of 2 L109
DMOBA/DSPC/Cholesterol/2KPEG-Chol, 50/20/28/2 N/P ratio of 2 L110
DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 1.5 L111
DMOBA/Cholesterol/2KPEG-DMG, N/P 67/30/3 ratio of 1.5 L112
DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/45/3 of 1.5 L113
DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 67/30/3 of 1.5 L114
DMOBA/Cholesterol/2KPEG-DMG, N/P 52/45/3 ratio of 2 L115
DMOBA/Cholesterol/2KPEG-DMG, N/P 67/30/3 ratio of 2 L116
DMLBA/Cholesterol/2KPEG-DMG, N/Pratio 52/45/3 of 2 L117
DMLBA/Cholesterol/2KPEG-DMG, N/P ratio 52/45/3 of 2 L118
LinCDMA/DSPC/Cholesterol/2KPEG- 43/38/10/2/7 DMG/Linoleyl alcohol,
N/P ratio of 2.85 L121 CLIM/DSPC/Cholesterol/2KPEG-DMG/, 48/40/10/2
N/P ratio of 3 L122 CLIM/Cholesterol/2KPEG-DMG/, N/P ratio 68/30/2
of 3 L123 CLinDMA/DSPC/Cholesterol/2KPEG- 43/38/10/3/7 DMG/Linoleyl
alcohol, N/P ratio of 2.85 L124 CLinDMA/DSPC/Cholesterol/2KPEG-
43/36/10/4/7 DMG/Linoleyl alcohol, N/P ratio of 2.85 L130
CLinDMA/DOPC/Chol/PEG-n-DMG, 48/39/10/3 N/P ratio of 3 L131
DMLBA/Cholesterol/2KPEG-DMG, N/Pratio 52/43/5 of 3 L132
DMOBA/Cholesterol/2KPEG-DMG, N/Pratio 52/43/5 of 3 L133
CLinDMA/DOPC/Chol/PEG-n-DMG, 48/40/10/2 N/P ratio of 3 L134
CLinDMA/DOPC/Chol/PEG-n-DMG, 48/37/10/5 N/P ratio of 3 L149
COIM/DSPC/Cholesterol/2KPEG-DMG/, N/P 48/40/10/2 ratio of 3 L155
CLinDMA/DOPC/Cholesterol/2KPEG- 43/38/10/2/7 DMG/Linoleyl alcohol,
N/P ratio of 2.85 L156 CLinDMA/DOPC/Cholesterol/2KPEG-DMG,
45/43/10/2 N/P ratio of 2.85 L162
CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/P ratio of 2.5
L163 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/P ratio of 2
L164 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/43/10/2 N/P ratio of
2.25 L165 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/15/2 N/P ratio
of 2.25 L166 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/15/2 N/P
ratio of 2.5 L167 CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 40/43/15/2
N/P ratio of 2 L174 CLinDMA/DSPC/DOPC/Cholesterol/2KPEG-
43/9/27/10/4/7 DMG/Linoleyl alcohol, N/P ratio of 2.85 L175
CLinDMA/DSPC/DOPC/Cholesterol/2KPEG- 43/27/9/10/4/7 DMG/Linoleyl
alcohol, N/P ratio of 2.85 L176 CLinDMA/DOPC/Cholesterol/2KPEG-
43/36/10/4/7 DMG/Linoleyl alcohol, N/P ratio of 2.85 L180
CLinDMA/DOPC/Cholesterol/2KPEG- 43/36/10/4/7 DMG/Linoleyl alcohol,
N/P ratio of 2.25 L181 CLinDMA/DOPC/Cholesterol/2KPEG- 43/36/10/4/7
DMG/Linoleyl alcohol, N/P ratio of 2 L182
CLinDMA/DOPC/Cholesterol/2KPEG-DMG, 45/41/10/4 N/P ratio of 2.25
N/P ratio = Nitrogen:Phosphorous ratio between cationic lipid and
nucleic acid
[0888] The 2KPEG utilized is PEG2000, a polydispersion which can
typically vary from .about.1500 to .about.3000 Da (i.e., where
PEG(n) is about 33 to about 67, or on average .about.45).
##STR00074## ##STR00075## ##STR00076## ##STR00077##
TABLE-US-00006 TABLE V Cell Line Tissue Cell Type % RNA KD 6.12
spleen B lymphocyte hybrid LF2K = 50% LNP97 = 90% LNP98 = 92% Raw
264.7 tumor macrophage/monocyte LF2K = 85% LNP54 = 75% LPN98 = 75%
MM14.Lu normal lung endothelial/epithelial LF2K = 90% LNP98 = 98%
NIH 3T3 embryo fibroblast LF2K = 95% LNP51 = 65% LPN54 = 65% LPN98
= 85% N/A lung primary macrophage LF2K = 50% LNP98 = 65%
TABLE-US-00007 TABLE VI LNP PROCESS FLOW CHART ##STR00078## (*siNA
2 is optional, shown for input into LNP siNA cocktail formulation,
additional siNA duplexes, e.g., siNA 3, siNA 4, siNA 5 etc. can be
used for siNA cocktails)
Sequence CWU 1
1
11121DNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 1cugauagggu gcuugcgagt t 21221DNAArtificial
SequenceSynthetic Target Sequence/siNA antisense region 2cucgcaagca
cccuaucagt t 21321DNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 3cctgtattcc catcccatcg t
21421DNAArtificial SequenceSynthetic Forward primer 4tgagccaaga
gaaacggact g 21527DNAArtificial SequenceSynthetic Reverse primer
5ttcgcaaaat acctatggga gtgggcc 27621DNAArtificial SequenceSynthetic
Probe FAM 6cugguacaga ccauauugat t 21721DNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 7ucaauauggu
cuguaccagt t 21821DNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 8ucagcauuac caagauuaat t
21921DNAArtificial SequenceSynthetic Target Sequence/siNA sense
region 9uuaaucuugg uaaugcugat t 211021DNAArtificial
SequenceSynthetic Target Sequence/siNA sense region 10accgugugaa
ucauugucut t 211121DNAArtificial SequenceSynthetic Target
Sequence/siNA sense region 11agacaaugau ucacacggut t 21
* * * * *