U.S. patent application number 13/168543 was filed with the patent office on 2012-03-08 for lipid encapsulated interfering rna.
This patent application is currently assigned to Protiva Biotherapeutics, Inc.. Invention is credited to Ellen Grace Ambegia, James Heyes, Ian MacLachlan.
Application Number | 20120058188 13/168543 |
Document ID | / |
Family ID | 34084527 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120058188 |
Kind Code |
A1 |
MacLachlan; Ian ; et
al. |
March 8, 2012 |
LIPID ENCAPSULATED INTERFERING RNA
Abstract
The present invention provides compositions and methods for
silencing gene expression by delivering nucleic acid-lipid
particles comprising a siRNA molecule to a cell.
Inventors: |
MacLachlan; Ian; (Mission,
CA) ; Ambegia; Ellen Grace; (Vancouver, CA) ;
Heyes; James; (Vancouver, CA) |
Assignee: |
Protiva Biotherapeutics,
Inc.
Burnaby
CA
|
Family ID: |
34084527 |
Appl. No.: |
13/168543 |
Filed: |
June 24, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11426907 |
Jun 27, 2006 |
7982027 |
|
|
13168543 |
|
|
|
|
10893121 |
Jul 16, 2004 |
|
|
|
11426907 |
|
|
|
|
60529406 |
Dec 11, 2003 |
|
|
|
60503279 |
Sep 15, 2003 |
|
|
|
60488144 |
Jul 16, 2003 |
|
|
|
Current U.S.
Class: |
424/489 ;
424/400; 435/375; 514/44A; 977/773; 977/800; 977/906 |
Current CPC
Class: |
A61P 31/20 20180101;
A61P 31/14 20180101; A61P 1/16 20180101; A61K 9/1272 20130101; A61P
3/06 20180101 |
Class at
Publication: |
424/489 ;
424/400; 435/375; 514/44.A; 977/773; 977/800; 977/906 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C12N 5/00 20060101 C12N005/00; A61K 9/51 20060101
A61K009/51; A61P 1/16 20060101 A61P001/16; A61P 31/14 20060101
A61P031/14; A61P 3/06 20060101 A61P003/06; A61P 31/20 20060101
A61P031/20; A61K 9/14 20060101 A61K009/14; A61K 31/7105 20060101
A61K031/7105 |
Claims
1-51. (canceled)
52. A nucleic acid-lipid particle, said nucleic acid-lipid particle
comprising: an interfering RNA; a cationic lipid; a non-cationic
lipid; and a conjugated lipid that inhibits aggregation of
particles.
53. The nucleic acid-lipid particle of claim 52, wherein said
interfering RNA is fully encapsulated in said nucleic acid-lipid
particle.
54. The nucleic acid-lipid particle of claim 52, wherein said
interfering RNA silences the expression of a gene selected from the
group consisting of a gene associated with viral infection and
survival, a gene associated with a metabolic disease or disorder,
and a gene associated with tumorigenesis and cell
transformation.
55. The nucleic acid-lipid particle of claim 52, wherein said
interfering RNA comprises an siRNA.
56. The nucleic acid-lipid particle of claim 55, wherein said siRNA
is about 19 to about 25 base pairs in length.
57. The nucleic acid-lipid particle of claim 55, wherein said siRNA
comprises 3' overhangs.
58. The nucleic acid-lipid particle of claim 55, wherein said siRNA
comprises 2'-O-methyl ribonucleotides.
59. The nucleic acid-lipid particle of claim 52, wherein the
conjugated lipid that inhibits aggregation of particles comprises a
polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid
conjugate, or combinations thereof.
60. The nucleic acid-lipid particle of claim 52, wherein the
conjugated lipid that inhibits aggregation of particles comprises a
PEG-diacylglycerol (DAG) conjugate, a PEG-dialkyloxypropyl (DAA)
conjugate, a PEG-phospholipid conjugate, a PEG-ceramide conjugate,
or combinations thereof.
61. The nucleic acid-lipid particle of claim 60, wherein the
PEG-DAA conjugate is selected from the group consisting of a
PEG-dilauryloxypropyl (C.sub.12), a PEG-dimyristyloxypropyl
(C.sub.14), a PEG-dipalmityloxypropyl (C.sub.16), a
PEG-distearyloxypropyl (C.sub.18), and combinations thereof.
62. The nucleic acid-lipid particle of claim 52, wherein said
non-cationic lipid comprises a phospholipid and cholesterol.
63. The nucleic acid-lipid particle of claim 52, wherein said
particle has a median diameter of less than about 100 nm.
64. A pharmaceutical composition comprising a nucleic acid-lipid
particle of claim 52 and a pharmaceutically acceptable carrier.
65. A method of introducing an interfering RNA into a cell, said
method comprising contacting said cell with a nucleic acid-lipid
particle of claim 52.
66. A method for silencing the expression of a target sequence,
said method comprising administering to a mammalian subject a
therapeutically effective amount of a nucleic acid-lipid particle
of claim 52.
67. A method for the in vivo delivery of an interfering RNA, said
method comprising administering to a mammalian subject a nucleic
acid-lipid particle of claim 52.
68. A method for the in vivo delivery of an interfering RNA to a
liver cell, said method comprising administering to a mammalian
subject a nucleic acid-lipid particle of claim 52.
69. A method for treating a disease in a mammalian subject, said
method comprising administering to said mammalian subject a
therapeutically effective amount of a nucleic acid-lipid particle
of claim 52.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/426,907, filed Jun. 27, 2006, which
application is a continuation of U.S. patent application Ser. No.
10/893,121, filed Jul. 16, 2004, which application claims the
benefit of U.S. Provisional Patent Application Nos. 60/529,406,
filed Dec. 11, 2003; 60/503,279, filed Sep. 15, 2003, and
60/488,144, filed Jul. 16, 2003, the disclosures of each of which
are hereby incorporated by reference in their entirety for all
purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for the therapeutic delivery of a nucleic acid by delivering a
serum-stable lipid delivery vehicle encapsulating the nucleic acid
to provide efficient RNA interference (RNAi) in a cell or mammal.
More particularly, the present invention is directed to using a
small interfering RNA (siRNA) encapsulated in a serum-stable lipid
particle having a small diameter suitable for systemic
delivery.
BACKGROUND OF THE INVENTION
[0003] RNA interference (RNAi) is an evolutionarily conserved,
sequence specific mechanism triggered by double stranded RNA
(dsRNA) that induces degradation of complementary target single
stranded mRNA and "silencing" of the corresponding translated
sequences (McManus and Sharp, Nature Rev. Genet. 3:737 (2002)).
RNAi functions by enzymatic cleavage of longer dsRNA strands into
biologically active "short-interfering RNA" (siRNA) sequences of
about 21-23 nucleotides in length (Elbashir, et al., Genes Dev.
15:188 (2001)). siRNA can be used downregulate or silence the
translation of a gene product of interest. For example, it is
desirable to downregulate genes associated with various diseases
and disorders.
[0004] Delivery of siRNA remains problematic (see, e.g., Novina and
Sharp, Nature 430:161-163 (2004); and Garber, J. Natl. Cancer Inst.
95 (7):500-2 (2003)). An effective and safe nucleic acid delivery
system is required for siRNA to be therapeutically useful. Naked
dsRNA administered to most subjects will: (1) be degraded by
endogenous nucleases; and (2) will not be able to cross cell
membranes to contact and silence their target gene sequences.
[0005] Viral vectors are relatively efficient gene delivery
systems, but suffer from a variety of safety concerns, such as
potential for undesired immune responses. Furthermore, viral
systems are rapidly cleared from the circulation, limiting
transfection to "first-pass" organs such as the lungs, liver, and
spleen. In addition, these systems induce immune responses that
compromise delivery with subsequent injections. As a result,
nonviral gene delivery systems are receiving increasing attention
(Worgall, et al., Human Gene Therapy 8:37 (1997); Peeters, et al.,
Human Gene Therapy 7:1693 (1996); Yei, et al., Gene Therapy 1: 192
(1994); Hope, et al., Molecular Membrane Biology 15:1 (1998)).
[0006] Plasmid DNA-cationic liposome complexes are currently the
most commonly employed nonviral gene delivery vehicles (Felgner,
Scientific American 276:102 (1997); Chonn, et al., Current Opinion
in Biotechnology 6:698 (1995)). For instance, cationic liposome
complexes made of an amphipathic compound, a neutral lipid, and a
detergent for transfecting insect cells are disclosed in U.S. Pat.
No. 6,458,382. Cationic liposome complexes are also disclosed in
U.S. Patent Application Publication No. 2003/0073640. Cationic
liposome complexes, however, are large, poorly defined systems that
are not suited for systemic applications and can elicit
considerable toxic side effects (Harrison, et al., Biotechniques
19:816 (1995); Li, et al., The Gene 4:891 (1997); Tam, et al, Gene
Ther. 7:1867 (2000)). As large, positively charged aggregates,
lipoplexes are rapidly cleared when administered in vivo, with
highest expression levels observed in first-pass organs,
particularly the lungs (Huang, et al., Nature Biotechnology 15:620
(1997); Templeton, et al., Nature Biotechnology 15:647 (1997);
Hofland, et al., Pharmaceutical Research 14:742 (1997)).
[0007] Other liposomal delivery systems include, for example, the
use of reverse micelles, anionic and polymer liposomes as disclosed
in, e.g., U.S. Pat. No. 6,429,200; U.S. Patent Application No.
2003/0026831; and U.S. Patent Application Nos. 2002/0081736 and
2003/0082103, respectively.
[0008] Recent work has shown that nucleic acids can be encapsulated
in small (about 70 nm diameter) "stabilized plasmid-lipid
particles" (SPLP) that consist of a single plasmid encapsulated
within a bilayer lipid vesicle (Wheeler, et al., Gene Therapy 6:271
(1999)). These SPLPs typically contain the "fusogenic" lipid
dioleoylphosphatidylethanolamine (DOPE), low levels of cationic
lipid (i.e., 10% or less), and are stabilized in aqueous media by
the presence of a poly(ethylene glycol) (PEG) coating. SPLP have
systemic application as they exhibit extended circulation lifetimes
following intravenous (i.v.) injection, accumulate preferentially
at distal tumor sites due to the enhanced vascular permeability in
such regions, and can mediate transgene expression at these tumor
sites. The levels of transgene expression observed at the tumor
site following i.v. injection of SPLP containing the luciferase
marker gene are superior to the levels that can be achieved
employing plasmid DNA-cationic liposome complexes (lipoplexes) or
naked DNA.
[0009] However, there remains a strong need in the art for novel
and more efficient methods and compositions for introducing nucleic
acids, such as siRNA, into cells. The present invention addresses
this and other needs.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides stable nucleic acid-lipid
particles (SNALP) useful for encapsulating one or more siRNA
molecules, methods of making SNALPs comprising siRNA, SNALPs
comprising siRNA and methods of delivering and/or administering the
SNALPs to a subject to silence expression of a target gene
sequence.
[0011] In one embodiment, the invention provide nucleic acid-lipid
particles comprising: a cationic lipid, a non-cationic lipid, a
conjugated lipid that inhibits aggregation of particles and a
siRNA. In some embodiments, the siRNA molecule is fully
encapsulated within the lipid bilayer of the nucleic acid-lipid
particle such that the nucleic acid in the nucleic acid-lipid
particle is resistant in aqueous solution to degradation by a
nuclease. The nucleic acid particle are substantially non-toxic to
mammals. The siRNA molecule may comprise about 15 to about 60
nucleotides. The siRNA molecule may be derived from a
double-stranded RNA greater than about 25 nucleotides in length. In
some embodiments the siRNA is transcribed from a plasmid, in
particular a plasmid comprising a DNA template of a target
sequence.
[0012] The cationic lipid may be one or more of
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), and N,N-dimethyl-2,3-dioleyloxy)propylamine
(DODMA), and a mixture thereof. The non-cationic lipid may be one
or more of dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine
(EPC), distearoylphosphatidylcholine (DSPC), cholesterol, and
combinations thereof.
[0013] The conjugated lipid that inhibits aggregation of particles
may be one or more of a polyethyleneglycol (PEG)-lipid conjugate; a
polyamide (ATTA)-lipid conjugate, and combinations thereof. The
PEG-lipid conjugate may be one or more of a PEG-dialkyloxypropyl
(DAA), a PEG-diacylglycerol (DAG), a PEG-phospholipid, a
PEG-ceramide, and combinations thereof. The PEG-DAG conjugate may
be one or more of a PEG-dilauroylglycerol (C.sub.12), a
PEG-dimyristoylglycerol (C.sub.14), a PEG-dipalmitoylglycerol
(C.sub.16), and a PEG-distearoylglycerol (C.sub.18), and
combinations thereof. The PEG-DAA conjugate may be one or more of a
PEG-dilauryloxypropyl (C.sub.12), a PEG-dimyristyloxypropyl (C14),
a PEG-dipalmityloxypropyl (C.sub.16), and a PEG-distearyloxypropyl
(C.sub.18), and combinations thereof. The nucleic acid-lipid
particle may further comprise a cationic polymer lipid.
[0014] In some embodiments, the particles are made by providing an
aqueous solution in a first reservoir and an organic lipid solution
in a second reservoir and mixing the aqueous solution with the
organic lipid solution so as to substantially instantaneously
produce a liposome encapsulating an interfering RNA. In some
embodiments, the particles are made by formation of hydrophobic
intermediate complexes in either detergent-based or organic
solvent-based systems, followed by removal of the detergent or
organic solvent. Preferred embodiments are charge-neutralized.
[0015] In one embodiment, the interfering RNA is transcribed from a
plasmid and the plasmid is combined with cationic lipids in a
detergent solution to provide a coated nucleic acid-lipid complex.
The complex is then contacted with non-cationic lipids to provide a
solution of detergent, a nucleic acid-lipid complex and
non-cationic lipids, and the detergent is then removed to provide a
solution, of serum-stable nucleic acid-lipid particles, in which
the plasmid comprising an interfering RNA template is encapsulated
in a lipid bilayer. The particles thus formed have a size of about
50-150 nm.
[0016] In another embodiment, serum-stable nucleic acid-lipid
particles are formed by preparing a mixture of cationic lipids and
non-cationic lipids in an organic solvent; contacting an aqueous
solution of nucleic acids comprising interfering RNA with the
mixture of cationic and non-cationic lipids to provide a clear
single phase; and removing the organic solvent to provide a
suspension of nucleic acid-lipid particles, in which the nucleic
acid is encapsulated in a lipid bilayer, and the particles are
stable in serum and have a size of about 50-150 nm.
[0017] The nucleic acid-lipid particles of the present invention
are useful for the therapeutic delivery of nucleic acids comprising
a siRNA sequence. In particular, it is an object of this invention
to provide in vitro and in vivo methods for treatment of a disease
in a mammal by downregulating or silencing the translation of a
target nucleic acid sequence. In these methods, a siRNA molecule is
formulated into a nucleic acid-lipid particle, and the particles
are administered to patients requiring such treatment (e.g., a
patient diagnosed with a disease or disorder associated with the
expression or overexpression of a gene comprising the target
nucleic acid sequence). Alternatively, cells are removed from a
patient, the siRNA is delivered in vitro, and the cells are
reinjected into the patient. In one embodiment, the present
invention provides for a method of introducing a siRNA molecule
into a cell by contacting a cell with a nucleic acid-lipid particle
comprising of a cationic lipid, a non-cationic lipid, a conjugated
lipid that inhibits aggregation, and a siRNA.
[0018] The nucleic acid-lipid particle may be administered, e.g.,
intravenously, parenterally or intraperitoneally. In one
embodiment, at least about 10% of the total administered dose of
the nucleic acid-lipid particles is present in plasma about 24, 36,
48, 60, 72, 84, or 96 hours after injection. In other embodiments,
more than 20%, 30%, 40% and as much as 60%, 70% or 80% of the total
injected dose of the nucleic acid-lipid particles is present in
plasma 24, 36, 48, 60, 72, 84, or 96 hours after injection. In one
embodiment, the presence of a siRNA in cells in a target tissue
(i.e., lung, liver, tumor or at a site of inflammation) is
detectable at 24, 48, 72 and 96 hours after administration. In one
embodiment, downregulation of expression of the target sequence is
detectable at 24, 48, 72 and 96 hours after administration. In one
embodiment, downregulation of expression of the target sequence
occurs preferentially in tumor cells or in cells at a site of
inflammation. In one embodiment, the presence of a siRNA in cells
at a site distal to the site of administration is detectable at
least four days after intravenous injection of the nucleic
acid-lipid particle. In another embodiment, the presence of a siRNA
in of cells in t a target tissue (i.e., lung, liver, tumor or at a
site of inflammation) is detectable at least four days after
injection of the nucleic acid-lipid particle.
[0019] The particles are suitable for use in intravenous nucleic
acid transfer as they are stable in circulation, of a size required
for pharmacodynamic behavior resulting in access to extravascular
sites and target cell populations. The invention also provides for
pharmaceutically acceptable compositions comprising a nucleic
acid-lipid particle.
[0020] Another embodiment of the present invention provides methods
for in vivo delivery of siRNA. A nucleic acid-lipid particle
comprising a cationic lipid, a non-cationic lipid, a conjugated
lipid that inhibits aggregation of particles, and siRNA is
administered (e.g., intravenously, subcutaneously,
intraperitoneally, or subdermally) to a subject (e.g., a mammal
such as a human). In some embodiments, the invention provides
methods for in vivo delivery of interfering RNA to the liver of a
mammalian subject.
[0021] A further embodiment of the present invention provides a
method of treating a disease or disorder in a mammalian subject. A
therapeutically effective amount of a nucleic acid-lipid particle
comprising a cationic lipid, a non-cationic lipid, a conjugated
lipid that inhibits aggregation of particles, and siRNA is
administered to the mammalian subject (e.g., a rodent such as a
mouse, a primate such as a human or a monkey) with the disease or
disorder. In some embodiments, the disease or disorder is
associated with expression and/or overexpression of a gene and
expression or overexpression of the gene is silenced by the siRNA.
In some embodiments, the disease is a viral disease such as, for
example, hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C,
Hepatitis D, Hepatitis E, Hepatitis G, or a combination thereof).
In some embodiment, the disease or disorder is a liver disease or
disorder, such as, for example, dyslipidemia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 illustrates downregulating .beta.-galactosidase
expression in CT26.CL25 cells via in vitro delivery of encapsulated
anti-.beta.-galactosidase siRNA in DSPC:Cholesterol:DODMA:PEG-DMG
liposomes.
[0023] FIG. 2 illustrates the structures of PEG-Diacylglycerols and
PEG-Ceramide C.sub.20.
[0024] FIG. 3 illustrates that clearance studies with LUVs showed
that SNALPs containing PEG-DAGs were comparable to SNALPs
containing PEG-CeramideC20.
[0025] FIG. 4 illustrates that SNALPs containing PEG-DAGs can be
formulated via a detergent dialysis method.
[0026] FIG. 5 illustrates the pharmacokinetic properties of SNALPs
containing PEG-DAGs.
[0027] FIG. 6 illustrates the biodistribution properties of SNALPs
containing PEG-DAGs.
[0028] FIG. 7 illustrates the luciferase gene expression 24 hrs
post IV administration of SPLPs containing PEG-CeramideC.sub.20
versus PEG-DAGs in Neuro-2a Tumor Bearing Male A/J Mice.
[0029] FIG. 8 illustrates the luciferase gene expression 48 hrs
post IV administration of SPLPs containing PEG-CeramideC.sub.20
versus PEG-DAGs in Neuro-2a Tumor Bearing Male A/J Mice.
[0030] FIG. 9 illustrates the luciferase gene expression 72 hrs
post IV administration of SPLPs containing PEG-CeramideC.sub.20
versus PEG-DAGs in Neuro-2a Tumor Bearing Male A/J Mice.
[0031] FIG. 10 illustrates the structures of three exemplary
PEG-dialkyloxypropyl derivatives suitable for use in the present
invention, i.e., N-(2,3-dimyristyloxypropyl) carbamate PEG.sub.2000
methyl ether PEG-C-DMA), N-(2,3-dimyristyloxypropyl) amide
PEG.sub.2000 methyl ether (i.e., PEG-A-DMA), and
N-(2,3-dimyristyloxypropyl) succinamide PEG.sub.2000 methyl ether
(i.e., PEG-S-DMA).
[0032] FIG. 11 illustrates data showing luciferase gene expression
in tumors 48 hours after intravenous administration of SPLP
comprising PEG-DAA conjugates and PEG-DAG conjugates.
[0033] FIG. 12 illustrates data showing luciferase gene expression
in liver, lung, spleen, heart, and tumor following intravenous
administration of SPLP comprising PEG-DAA conjugates and PEG-DAG
conjugates.
[0034] FIG. 13 illustrates data from clearance studies in Neuro-2a
tumor bearing male A/J mice after administration of SPLPs
comprising a PEG-DAA conjugate and containing a plasmid encoding
luciferase under the control of the CMV promoter and SNALPs
comprising a PEG-DAA conjugate and containing anti-luciferase
siRNA.
[0035] FIG. 14 illustrates data from studies of the pharmacokinetic
properties of SPLPs comprising a PEG-DAA conjugate and containing a
plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA in Neuro-2a tumor bearing male A/J mice.
[0036] FIG. 15 illustrates data from clearance studies in Neuro-2a
tumor bearing male A/J mice after administration of SPLPs
comprising a PEG-DAA conjugate or a PEG-DAG conjugate and
containing a plasmid encoding luciferase under the control of the
CMV promoter, pSPLPs comprising a PEG-DAG conjugate and containing
a plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA.
[0037] FIG. 16 illustrates data from studies of the pharmacokinetic
properties of SPLPs comprising a PEG-DAA conjugate or a PEG-DAG
conjugate and containing a plasmid encoding luciferase under the
control of the CMV promoter, pSPLPs comprising a PEG-DAG conjugate
and containing a plasmid encoding luciferase under the control of
the CMV promoter and SNALPs comprising a PEG-DAA conjugate and
containing anti-luciferase siRNA in Neuro-2a tumor bearing male A/J
mice.
[0038] FIG. 17 illustrates in vitro data demonstrating silencing of
luciferase expression in luciferase expressing cells treated with
SPLPs comprising a PEG-lipid conjugate and containing a plasmid
encoding luciferase under the control of the CMV promoter and
SNALPs comprising a PEG-lipid conjugate conjugate and containing
anti-luciferase siRNA.
[0039] FIG. 18 illustrates in vivo data demonstrating silencing of
luciferase expression in Neuro-2a tumor bearing male Al mice
treated with SPLPs comprising a PEG-DAA conjugate and containing a
plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA.
[0040] FIG. 19 illustrates in vivo data demonstrating silencing of
luciferase expression in Neuro-2a tumor bearing male A/J mice
treated with SPLPs comprising a PEG-DAA conjugate and containing a
plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA.
[0041] FIG. 20 illustrates in vivo data demonstrating silencing of
luciferase expression in Neuro-2a tumor bearing male A/J mice
treated with SPLPs comprising a PEG-DAA conjugate and containing a
plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA.
[0042] FIG. 21 illustrates in vivo data demonstrating silencing of
luciferase expression in Neuro-2a tumor bearing male A/J mice
treated with SPLPs comprising a PEG-DAA conjugate and containing a
plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA.
[0043] FIG. 22 illustrates in vivo data demonstrating silencing of
luciferase expression in Neuro-2a tumor bearing male A/J mice
treated with SPLPs comprising a PEG-DAA conjugate and containing a
plasmid encoding luciferase under the control of the CMV promoter
and SNALPs comprising a PEG-DAA conjugate and containing
anti-luciferase siRNA.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0044] The present invention provides stable nucleic acid-lipid
particles (SNALP) useful for encapsulating one or more siRNA
molecules, methods of making SNALPs comprising siRNA, SNALPs
comprising siRNA and methods of delivering and/or administering the
SNALPs to a subject to silence expression of a target gene
sequence.
[0045] The present invention is based on the unexpected success of
encapsulating short interfering RNA (siRNA) molecules in SNALPs.
Using the methods of the present invention, siRNA molecules are
encapsulated in SNALPs with an efficiency greater than 70%, more
usually with an efficiency greater than 80 to 90%. The SNALPs
described herein can conveniently be used in vitro and in vivo to
efficiently deliver administer siRNA molecules locally or
systemically to cells expressing a target gene. Once delivered, the
siRNA molecules in the SNALPs silence expression of the target
gene.
[0046] The SNALPs described herein are typically <150 nm
diameter and remain intact in the circulation for an extended
period of time in order to achieve delivery of siRNA to target
tissues. The SNALPs are highly stable, serum-resistant nucleic
acid-containing particles that does not interact with cells and
other components of the vascular compartment. Moreover, the SNALPs
also readily interact with target cells at a disease site in order
to facilitate intracellular delivery of a desired nucleic acid
(e.g., a siRNA or a plasmid encoding a siRNA).
II. Definitions
[0047] The term "lipid" refers to a group of organic compounds that
include, but are not limited to, esters of fatty acids and are
characterized by being insoluble in water, but soluble in many
organic solvents. They are usually divided into at least three
classes: (1) "simple lipids which include fats and oils as well as
waxes; (2) "compound lipids" which include phospholipids and
glycolipids; (3) "derived lipids" such as steroids.
[0048] "Lipid vesicle" refers to any lipid composition that can be
used to deliver a compound including, but not limited to,
liposomes, wherein an aqueous volume is encapsulated by an
amphipathic lipid bilayer; or wherein the lipids coat an interior
comprising a large molecular component, such as a plasmid
comprising an interfering RNA sequence, with a reduced aqueous
interior; or lipid aggregates or micelles, wherein the encapsulated
component is contained within a relatively disordered lipid
mixture.
[0049] As used herein, "lipid encapsulated" can refer to a lipid
formulation that provides a compound with full encapsulation,
partial encapsulation, or both. In a preferred embodiment, the
nucleic acid is fully encapsulated in the lipid formulation.
[0050] As used herein, the term "SNALP" refers to a stable nucleic
acid lipid particle. A SNALP represents a vesicle of lipids coating
a reduced aqueous interior comprising a nucleic acid such as an
interfering RNA sequence or a plasmid from which an interfering RNA
is transcribed.
[0051] The term "vesicle-forming lipid" is intended to include any
amphipathic lipid having a hydrophobic moiety and a polar head
group, and which by itself can form spontaneously into bilayer
vesicles in water, as exemplified by most phospholipids.
[0052] The term "vesicle-adopting lipid" is intended to include any
amphipathic lipid that is stably incorporated into lipid bilayers
in combination with other amphipathic lipids, with its hydrophobic
moiety in contact with the inferior, hydrophobic region of the
bilayer membrane, and its polar head group moiety oriented toward
the exterior, polar surface of the membrane. Vesicle-adopting
lipids include lipids that on their own tend to adopt a nonlamellar
phase, yet which are capable of assuming a bilayer structure in the
presence of a bilayer-stabilizing component. A typical example is
DOPE (dioleoylphosphatidylethanolamine). Bilayer stabilizing
components include, but are not limited to, conjugated lipids that
inhibit aggregation of the SNALPs, polyimide oligomers (e.g.,
ATTA-lipid derivatives), peptides, proteins, detergents,
lipid-derivatives, PEG-lipid derivatives such as PEG coupled to
dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to
phosphatidylethanolamines, and PEG conjugated to ceramides (see,
U.S. Pat. No. 5,885,613, which is incorporated herein by
reference).
[0053] The term "amphipathic lipid" refers, in part, to any
suitable material wherein the hydrophobic portion of the lipid
material orients into a hydrophobic phase, while the hydrophilic
portion orients toward the aqueous phase. Amphipathic lipids are
usually the major component of a lipid vesicle. Hydrophilic
characteristics derive from the presence of polar or charged groups
such as carbohydrates, phosphate, carboxylic, sulfato, amino,
sulthydryl, nitro, hydroxy and other like groups. Hydrophobicity
can be conferred by the inclusion of apolar groups that include,
but are not limited to, long chain saturated and unsaturated
aliphatic hydrocarbon groups and such groups substituted by one or
more aromatic, cycloaliphatic or heterocyclic group(s). Examples of
amphipathic compounds include, but are not limited to,
phospholipids, aminolipids and sphingolipids. Representative
examples of phospholipids include, but are not limited to,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine or
dilinoleoylphosphatidylcholine. Other compounds lacking in
phosphorus, such as sphingolipid, glycosphingolipid families,
diacylglycerols and .beta.acyloxyacids, are also within the group
designated as amphipathic lipids. Additionally, the amphipathic
lipid described above can be mixed with other lipids including
triglycerides and sterols.
[0054] The term "neutral lipid" refers to any of a number of lipid
species that exist either in an uncharged or neutral zwitterionic
form at a selected pH. At physiological pH, such lipids include,
for example, diacylphosphatidylcholine,
diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,
cholesterol, cerebrosides and diacylglycerols.
[0055] The term "noncationic lipid" refers to any neutral lipid as
described above as well as anionic lipids.
[0056] The term "anionic lipid" refers to any lipid that is
negatively charged at physiological pH. These lipids include, but
are not limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanolamines, N-succinyl phosphatidylethanolamines,
N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,
palmitoyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
[0057] The term "cationic lipid" refers to any of a number of lipid
species that carry a net positive charge at a selected pH, such as
physiological pH. Such lipids include, but are not limited to,
N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTMA"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP"); 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol
("DC-Chol") and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). The following lipids are cationic and have a
positive charge at below physiological pH: DODAP, DODMA, DMDMA and
the like.
[0058] The term "hydrophobic lipid" refers to compounds having
apolar groups that include, but are not limited to, long chain
saturated and unsaturated aliphatic hydrocarbon groups and such
groups optionally substituted by one or more aromatic,
cycloaliphatic or heterocyclic group(s). Suitable examples include,
but are not limited to, diacylglycerol, dialkylglycerol,
N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and
1,2-dialkyl-3-aminopropane.
[0059] The term "fusogenic" refers to the ability of a liposome, an
SNALP or other drug delivery system to fuse with membranes of a
cell. The membranes can be either the plasma membrane or membranes
surrounding organelles, e.g., endosome, nucleus, etc.
[0060] The term "diacylglycerol" refers to a compound having
2-fatty acyl chains, R.sup.1 and R.sup.2, both of which have
independently between 2 and 30 carbons bonded to the 1- and
2-position of glycerol by ester linkages. The acyl groups can be
saturated or have varying degrees of unsaturation. Diacylglycerols
have the following general formula:
##STR00001##
[0061] The term "dialkyloxypropyl" refers to a compound having
2-alkyl chains, R.sup.1 and R.sup.2, both of which have
independently between 2 and 30 carbons. The alkyl groups can be
saturated or have varying degrees of unsaturation.
Dialkyloxypropyls have the following general formula:
##STR00002##
The term "ATTA" or "polyamide" refers to, but is not limited to,
compounds disclosed in U.S. Pat. Nos. 6,320,017 and 6,586,559, both
of which are incorporated herein by reference. These compounds
include a compound having the formula
##STR00003##
wherein: R is a member selected from the group consisting of
hydrogen, alkyl and acyl; R.sup.1 is a member selected from the
group consisting of hydrogen and alkyl; or optionally, R and
R.sup.1 and the nitrogen to which they are bound form an azido
moiety; R.sup.2 is a member of the group selected from hydrogen,
optionally substituted alkyl, optionally substituted aryl and a
side chain of an amino acid; R.sup.3 is a member selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto,
hydrazino, amino and NR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5
are independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p
is 1 to 4; and q is 0 or 1. It will be apparent to those of skill
in the art that other polyamides can be used in the compounds of
the present invention.
[0062] The term "nucleic acid" or "polynucleotide" refers to a
polymer containing at least two deoxyribonucleotides or
ribonucleotides in either single- or double-stranded form. Unless
specifically limited, the terms encompasses nucleic acids
containing known analogues of natural nucleotides that have similar
binding properties as the reference nucleic acid and are
metabolized in a manner similar to naturally occurring nucleotides.
Unless otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions), alleles, orthologs, SNPs,
and complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell.
Probes 8:91-98 (1994)). "Nucleotides" contain a sugar deoxyribose
(DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides
are linked together through the phosphate groups. "Bases" include
purines and pyrimidines, which further include natural compounds
adenine, thymine, guanine, cytosine, uracil, inosine, and natural
analogs, and synthetic derivatives of purines and pyrimidines,
which include, but are not limited to, modifications which place
new reactive groups such as, but not limited to, amines, alcohols,
thiols, carboxylates, and alkylhalides. DNA may be in the form of
antisense, plasmid DNA, parts of a plasmid DNA, pre-condensed DNA,
product of a polymerase chain reaction (PCR), vectors (P1, PAC,
BAC, YAC, artificial chromosomes), expression cassettes, chimeric
sequences, chromosomal DNA, or derivatives of these groups. The
term nucleic acid is used interchangeably with gene, cDNA, mRNA
encoded by a gene, and an interfering RNA molecule.
[0063] The term "gene" refers to a nucleic acid (e.g., DNA or RNA)
sequence that comprises partial length or entire length coding
sequences necessary for the production of a polypeptide or a
polypeptide precursor (e.g., polypeptides or polypeptide preursors
from hepatitis virus A, B, C, D, E, or G; or herpes simplex
virus).
[0064] "Gene product," as used herein, refers to a product of a
gene such as an RNA transcript, including, e.g., mRNA.
[0065] The term "interfering RNA" or "RNAi" or "interfering RNA
sequence" refers to double-stranded RNA (i.e., duplex RNA) that is
capable of reducing or inhibiting expression of a target gene
(i.e., by mediating the degradation of mRNAs which are
complementary to the sequence of the interfering RNA) when the
interfering RNA is in the same cell as the target gene. Interfering
RNA thus refers to the double stranded RNA formed by two
complementary strands or by a single, self-complementary strand.
Interfering RNA typically has substantial or complete identity to
the target gene. The sequence of the interfering RNA can correspond
to the full length target gene, or a subsequence thereof.
Interfering RNA includes small-interfering RNA" or "siRNA," i.e.,
interfering RNA of about 15-60, 15-50, 15-50, or 15-40 (duplex)
nucleotides in length, more typically about, 15-30, 15-25 or 19-25
(duplex) nucleotides in length, and is preferably about 20-24 or
about 21-22 or 21-23 (duplex) nucleotides in length (e.g., each
complementary sequence of the double stranded siRNA is 15-60,
15-50, 15-50, 15-40, 15-30, 15-25 or 19-25 nucleotides in length,
preferably about 20-24 or about 21-22 or 21-23 nucleotides in
length, and the double stranded siRNA is about 15-60, 15-50, 15-50,
15-40, 15-30, 15-25 or 19-25 preferably about 20-24 or about 21-22
or 21-23 base pairs in length). siRNA duplexes may comprise 3'
overhangs of about 1 to about 4 nucleotides, preferably of about 2
to about 3 nucleotides and 5' phosphate termini. The siRNA can be
chemically synthesized or may be encoded by a plasmid (e.g.,
transcribed as sequences that automatically fold into duplexes with
hairpin loops). siRNA can also be generated by cleavage of longer
dsRNA (e.g., dsRNA greater than about 25 nucleotides in length)
with the E coli RNase III or Dicer. These enzymes process the dsRNA
into biologically active siRNA (see, e.g., Yang et al., PNAS USA
99: 9942-7 (2002); Calegari et al., PNAS USA 99: 14236 (2002);
Byrom et al., Ambion TechNotes 10 (1): 4-6 (2003); Kawasaki et al.,
Nucleic Acids Res. 31: 981-7 (2003); Knight and Bass, Science 293:
2269-71 (2001); and Robertson et al., J. Biol. Chem. 243: 82
(1968)). Preferably, dsRNA are at least 50 nucleotides to about
100, 200, 300, 400 or 500 nucleotides in length. A dsRNA may be as
long as 1000, 1500, 2000, 5000 nucleotides in length, or longer.
The dsRNA can encode for an entire gene transcript or a partial
gene transcript.
[0066] "Substantial identity" refers to a sequence that hybridizes
to a reference sequence under stringent conditions, or to a
sequence that has a specified percent identity over a specified
region of a reference sequence.
[0067] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization.
[0068] Exemplary stringent hybridization conditions can be as
following: 50% formamide, 5.times.SSC, and 1% SDS, incubating at
42.degree. C., or, 5.times.SSC, 1% SDS, incubating at 65.degree.
C., with wash in 0.2.times.SSC, and 0.1% SDS at 65.degree. C. For
PCR, a temperature of about 36 C is typical for low stringency
amplification, although annealing temperatures may vary between
about 32 C and 48 C depending on primer length. For high stringency
PCR amplification, a temperature of about 62 C is typical, although
high stringency annealing temperatures can range from about 50 C to
about 65 C, depending on the primer length and specificity. Typical
cycle conditions for both high and low stringency amplifications
include a denaturation phase of 90 C-95 C for 30 sec-2 min., an
annealing phase lasting 30 sec.-2 min., and an extension phase of
about 72 C for 1-2 min. Protocols and guidelines for low and high
stringency amplification reactions are provided, e.g., in Innis et
al. (1990) PCR Protocols, A Guide to Methods and Applications,
Academic Press, Inc. N.Y.).
[0069] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code., In
such cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide, conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al.
[0070] The terms "substantially identical" or "substantial
identity," in the context of two or more nucleic acids, refer to
two or more sequences or subsequences that are the same or have a
specified percentage of nucleotides that are the same (i.e., at
least about 60%, preferably 65%, 70%, 75%, preferably 80%, 85%,
90%, or 95% identity over a specified region), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
This definition, when the context indicates, also refers
analogously to the complement of a sequence. Preferably, the
substantial identity exists over a region that is at least about 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides in
length.
[0071] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0072] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment,of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0073] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0074] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0075] The phrase "inhibiting expression of a target gene" refers
to the ability of a siRNA of the invention to initiate gene
silencing of the target gene. To examine the extent of gene
silencing, samples or assays of the organism of interest or cells
in culture expressing a particular construct are compared to
control samples lacking expression of the construct. Control
samples (lacking construct expression) are assigned a relative
value of 100%. Inhibition of expression of a target gene
is,achieved when the test value relative to the control is about
90%, preferably 50%, more preferably 25-0%. Suitable assays
include, e.g., examination of protein or mRNA levels using
techniques known to those of skill in the art such as dot blots,
northern blots, in situ hybridization, ELISA, immunoprecipitation,
enzyme function, as well as phenotypic assays known to those of
skill in the art.
[0076] "Nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in single- or double-stranded
form. The term encompasses nucleic acids containing known
nucleotide analogs or modified backbone residues or linkages, which
are synthetic, naturally occurring, and non-naturally occurring,
which have similar binding properties as the reference nucleic
acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
[0077] By "silencing" or "downregulation" of a gene or nucleic acid
is intended to mean a detectable decrease of translation of a
target nucleic acid sequence, i.e., the sequence targeted by the
RNAi, or a decrease in the amount or activity of the target
sequence or protein, in comparison to the level that is detected in
the absence of the interfering RNA or other nucleic acid sequence.
A detectable decrease can be as small as about 5% or 10%, or as
great as about 80%, 90% or 100%. More typically, a detectable
decrease is about 20%, 30%, 40%, 50%, 60%, or 70%.
[0078] A "therapeutically effective amount" or an "effective
amount" of a siRNA is an amount sufficient to produce the desired
effect, e.g., a decrease in the expression of a target sequence in
comparison to the normal expression level detected in the absence
of the siRNA.
[0079] As used herein, the term "aqueous solution" refers to a
composition comprising in whole, or in part, water.
[0080] As used herein, the term "organic lipid solution" refers to
a composition comprising in whole, or in part, an organic solvent
having a lipid.
[0081] "Distal site," as used herein, refers to a physically
separated site, which is not limited to an adjacent capillary bed,
but includes sites broadly distributed throughout an organism. In
some embodiments, distal site refers to a site physically separated
from a disease site (e.g., the site of a tumor, the site of
inflammation, or the site of an infection).
[0082] "Serum-stable" in relation to nucleic acid-lipid particles
means that the particle is not significantly degraded after
exposure to a serum or nuclease assay that would significantly
degrade free DNA. Suitable assays include, for example, a standard
serum assay or a DNAse assay such as those described in the
Examples below.
[0083] "Systemic delivery," as used herein, refers to delivery that
leads to a broad biodistribution of a compound within an organism.
Some techniques of administration can lead to the systemic delivery
of certain compounds, but not others. Systemic delivery means that
a useful, preferably therapeutic, amount of a compound is exposed
to most parts of the body. To obtain broad biodistribution
generally requires a blood lifetime such that the compound is not
rapidly degraded or cleared (such as by first pass organs (liver,
lung, etc.) or by rapid, nonspecific cell binding) before reaching
a disease site distal to the site of administration. Systemic
delivery of nucleic acid-lipid particules can be by any means known
in the art including, for example, intravenous, subcutaneous,
intraperitoneal, In a preferred embodiment, systemic delivery of
nucleic acid-lipid particles is by intravenous delivery.
[0084] "Local delivery" as used herein refers to delivery of a
compound directly to a target site within an organism. For example,
a compound can be locally delivered by direct injection into a
disease site such as a tumor or other target site such as a site of
inflammation or a target organ such as the liver, heart, pancreas,
kidney, and the like,
III. Stable Nucleic Acid-Lipid Particles
[0085] The stable nucleic acid-lipid particles (SNALPs) described
herein typically comprise a nucleic acid (e.g., a siRNA sequence or
a DNA sequence encoding a siRNA sequence), a cationic lipid, a
noncationic lipid and a bilayer stabilizing component such as,
e.g., a conjugated lipid that inhibits aggregation of the SNALPs.
The SNALPs of the present invention have a mean diameter of less
than about 150 nm and are substantially nontoxic. In addition,
nucleic acids encapsulated in the SNALPs of the present invention
are resistant in aqueous solution to degradation with a
nuclease.
A. Cationic Lipids
[0086] Various suitable cationic lipids may be used in the SNALPs
described herein, either alone or in combination with one or more
other cationic lipid species or neutral lipid species.
[0087] Cationic lipids which are useful in the present invention
can be any of a number of lipid species which carry a net positive
charge at physiological pH, for example: DODAC, DOTMA, DDAB, DOTAP,
DOSPA, DOGS, DC-Chol and DMRIE, or combinations thereof. A number
of these lipids and related analogs, which are also useful in the
present invention, have been described in U.S. Pat. Nos. 5,208,036,
5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992, the
disclosures of each of which are incorporated herein by
reference.
[0088] The cationic lipid typically comprises from about 2% to
about 60% of the total lipid present in said particle, preferably
from about 5% to about 45% of the total lipid present in said
particle. In certain preferred embodiments, the cationic lipid
comprises from about 5% to about 15% of the total lipid present in
said particle. In other preferred embodiments, the cationic lipid
comprises from about 40% to about 50% of the total lipid present in
said particle. Depending on the intended use of the nucleic
acid-lipid particles, the proportions of the components are varied
and the delivery efficiency of a particular formulation can be
measured using an endosomal release parameter (ERP) assay. For
example, for systemic delivery, the cationic lipid may comprise
from about 5% to about 15% of the total lipid present in said
particle and for local or regional delivery, the cationic lipid
comprises from about 40% to about 50% of the total lipid present in
said particle.
B. Noncationic Lipids
[0089] The noncationic lipid component of the SNALPs described
herein 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),
palmitoyloleyolphosphatidylglycerol (POPG), dip
almitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-
phosphatidylethanolamine (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.,
stearylatnine, 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 U.S. Pat. No. 5,820,873, incorporated
herein by reference.
[0090] In preferred embodiments, the noncationic lipids are
diacylphosphatidylcholine (e.g., distearoylphosphatidylcholine,
dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine and
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 C.sub.10-C.sub.24 carbon
chains. More preferably the acyl groups are lauroyl, myristoyl,
palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the noncationic lipid will include one or more of
cholesterol, 1,2-sn-dioleoylphosphatidylethanolamine, or egg
sphingomyelin (ESM).
[0091] The non-cationic lipid typically comprises from about 5% to
about 90% of the total lipid present in said particle, preferably
from about 20% to about 85% of the total lipid present in said
particle. The PEG-DAG conjugate typically comprises from 1% to
about 20% of the total lipid present in said particle, preferably
from 4% to about 15% of the total lipid present in said particle.
The nucleic acid-lipid particles of the present invention may
further comprise cholesterol. If present, the cholesterol typically
comprises from about 10% to about 60% of the total lipid present in
said particle, preferably the cholesterol comprises from about 20%
to about 45% of the total lipid present in said particle.
C. Bilayer Stabilizing Component
[0092] In one embodiment, the SNALP further comprises a bilayer
stabilizing component (BSC). Suitable BSCs include, but are not
limited to, polyamide oligomers, peptides, proteins, detergents,
lipid-derivatives, PEG-lipids, such as PEG coupled to
dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol
(PEG-DAG), PEG coupled to phosphatidylethanolamine (PE) (PEG-PE),
or PEG conjugated to ceramides, or a mixture thereof (see, U.S.
Pat. No. 5,885,613, which is incorporated herein by reference). In
one embodiment, the bilayer stabilizing component is a PEG-lipid,
or an ATTA-lipid. In one preferred embodiment, the BSC is a
conjugated lipid that inhibits aggregation of the SNALPs. Suitable
conjugated lipids include, but are not limited to PEG-lipid
conjugates, ATTA-lipid conjugates, cationic-polymer-lipid
conjugates (CPLs) or mixtures thereof. In one preferred embodiment,
the SNALPs comprise either a PEG-lipid conjugate or an ATTA-lipid
conjugate together with a CPL.
[0093] Typically, the bilayer stabilizing component is present
ranging from about 0.5% to about 50% of the total lipid present in
the particle. In a preferred embodiment, the bilayer stabilizing
component is present from about 0.5% to about 25% of the total
lipid in the particle. In other preferred embodiments, the bilayer
stabilizing component is present from about 1% to about 20%, or
about 3% to about 15% or about 4% to about 10% of the total lipid
in the particle. One of ordinary skill in the art will appreciate
that the concentration of the bilayer stabilizing component can be
varied depending on the bilayer stabilizing component employed and
the rate at which the liposome is to become fusogenic.
[0094] By controlling the composition and concentration of the
bilayer stabilizing component, one can control the rate at which
the bilayer stabilizing component exchanges out of the liposome
and, in turn, the rate at which the liposome becomes fusogenic. For
instance, when a polyethyleneglycol-phosphatidylethanolamine
conjugate or a polyethyleneglycol-ceramide conjugate is used as the
bilayer stabilizing component, the rate at which the liposome
becomes fusogenic can be varied, for example, by varying the
concentration of the bilayer stabilizing component, by varying the
molecular weight of the polyethyleneglycol, or by varying the chain
length and degree of saturation of the acyl chain groups on the
phosphatidylethanolamine or the ceramide. In addition, other
variables including, for example, pH, temperature, ionic strength,
etc. can be used to vary and/or control the rate at which the
liposome becomes fusogenic. Other methods which can be used to
control the rate at which the liposome becomes fusogenic will
become apparent to those of skill in the art upon reading this
disclosure.
1. Diacylglycerol-Polyethyleneglycol Conjugates
[0095] In one embodiment, the bilayer stabilizing component
comprises a diacylglycerol-polyethyleneglycol conjugate, i.e., a
DAG-PEG conjugate or a PEG-DAG conjugate. In a preferred
embodiment, the DAG-PEG conjugate is a dilaurylglycerol
(C.sub.12)-PEG conjugate, dimyristylglycerol (C.sub.14)-PEG
conjugate (DMG), a dipalmitoylglycerol (C.sub.16)-PEG conjugate or
a distearylglycerol (C.sub.18)-PEG conjugate (DSG). Those of skill
in the art will readily appreciate that other diacylglycerols can
be used in the DAG-PEG conjugates of the present invention.
Suitable DAG-PEG conjugates for use in the present invention, and
methods of making and using them, are disclosed in U.S. application
Ser. No. 10/136,707 published as U.S.P.A. 2003/0077829, and PCT
Patent Application No. CA 02/00669, each of which is incorporated
herein in its entirety by reference.
2. Dialkyloxypropyl Conjugates
[0096] In another embodiment, the bilayer stabilizing component
comprises a dialkyloxypropyl conjugate, i.e., a PEG-DAA conjugate.
In one preferred embodiment, the PEG-DAA conjugate has the
following formula:
##STR00004##
[0097] In Formula I, R.sup.1 and R.sup.2 are independently selected
and are long-chain alkyl groups having from about 10 to about 22
carbon atoms. The long-chain alkyl groups can be saturated or
unsaturated. Suitable alkyl groups include, but are not limited to,
lauryl (C12), myristyl (C14), palmityl (C16), stearyl (C18) and
icosyl (C20). In preferred embodiments, R.sup.1 and R.sup.2 are the
same, i.e., R.sup.1 and R.sup.2 are both myristyl (i.e.,
dimyristyl), R.sup.1 and R.sup.2 are both stearyl (i.e.,
distearyl), etc. In Formula I, PEG is a polyethylene glycol having
an average molecular weight of from about 550 to about 8,500
daltons. In a preferred embodiment, the PEG has an average
molecular weight of from about 1000 to about 5000 daltons, more
preferably, from about 1,000 to about 3,000 daltons and, even more
preferably, of about 2,000 daltons. The PEG can be optionally,
substituted by an alkyl, alkoxy, acyl or aryl group. In Formula I,
L is a linker moiety. Any linker moiety suitable for coupling the
PEG to the dialkyloxypropyl backbone can be used. Suitable linker
moieties include, but are not limited to, amido (--C(O)NH--), amino
(--NR--), carbonyl (--C(O)--), carbonate (O--C(O)O--), carbamate
(--NHC(O)O--), urea (--NHC(O)NH--), succinyl
(--(O)CCH.sub.2CH.sub.2C(O)--), ether, disulphide, and combinations
thereof. Other suitable linkers are well known in the art.
[0098] Phosphatidylethanolamines having a variety of acyl chain
groups of varying chain lengths and degrees of saturation can be
conjugated to polyethyleneglycol to form the bilayer stabilizing
component. Such phosphatidylethanolamines are commercially
available, or can be isolated or synthesized using conventional
techniques known to those of skilled in the art.
Phosphatidylethanolamines containing saturated or unsaturated fatty
acids with carbon chain lengths in the range of C.sub.10 to
C.sub.20 are prefefred. Phosphatidylethanolamines with mono- or
diunsaturated fatty acids and mixtures of saturated and unsaturated
fatty acids can also be used. Suitable phosphatidylethanolamines
include, but are not limited to, the following:
dimyristoylphosphatidylethanolamine (DMPE),
dipalmitoylphosphatidylethanolamine (DPPE),
dioleoylphosphatidylethanolamine (DOPE) and
distearoylphosphatidylethanolamine (DSPE).
[0099] As with the phosphatidylethanolamines, ceramides having a
variety of acyl chain groups of varying chain lengths and degrees
of saturation can be coupled to polyethyleneglycol to form the
bilayer stabilizing component. It will be apparent to those of
skill in the art that in contrast to the phosphatidylethanolamines,
ceramides have only one acyl group which can be readily varied in
terms of its chain length and degree of saturation. Ceramides
suitable for use in accordance with the present invention are
commercially available. In addition, ceramides can be isolated, for
example, from egg or brain using well-known isolation techniques
or, alternatively, they can be synthesized using the methods and
techniques disclosed in U.S. Pat. No. 5,820,873, which is
incorporated herein by reference. Using the synthetic routes set
forth in the foregoing application, ceramides having saturated or
unsaturated fatty acids with carbon chain lengths in the range of
C.sub.2 to C.sub.31 can be prepared.
3. Cationic Polymer Lipids
[0100] Cationic polymer lipids (CPLs) can also be used in the
SNALPS described herein.
[0101] Suitable CPL typically have the following architectural
features: (1) a lipid anchor, such as a hydrophobic lipid, for
incorporating the CPLs into the lipid bilayer; (2) a hydrophilic
spacer, such as a polyethylene glycol, for linking the lipid anchor
to a cationic head group; and (3) a polycationic moiety, such as a
naturally occurring amino acid, to produce a protonizable cationic
head group. Suitable SNALPs and SNALP-CPLs for use in the present
invention, and methods of making and using SNALPs and SNALP-CPLs,
are disclosed, e.g., in U.S. application Ser. Nos. 09/553,639 and
09/839,707 (published as U.S.P.A. 2002/0072121) and PCT Patent
Application No. CA 00/00451 (published as WO 00/62813), each of
which is incorporated herein in its entirety by reference.
[0102] Briefly, the present invention provides a compound of
Formula II:
A-W-Y I
wherein A, W and Y are as follows.
[0103] With reference to Formula II, "A" is a lipid moiety such as
an amphipathic lipid, a neutral lipid or a hydrophobic lipid that
acts as a lipid anchor. Suitable lipid examples include
vesicle-forming lipids or vesicle adopting lipids and include, but
are not limited to, diacylglycerolyls, dialkylglycerolyls,
N-N-dialkylaminos, 1,2-diacyloxy-3-aminopropanes and
1,2-dialkyl-3-aminopropanes.
[0104] "W" is a polymer or an oligomer, such as a hydrophilic
polymer or oligomer. Preferably, the hydrophilic polymer is a
biocompatable polymer that is nonimmunogenic or possesses low
inherent immunogenicity. Alternatively, the hydrophilic polymer can
be weakly antigenic if used with appropriate adjuvants. Suitable
nonimmunogenic polymers include, but are not limited to, PEG,
polyamides, polylactic acid, polyglycolic acid, polylactic
acid/polyglycolic acid copolymers and combinations thereof. In a
preferred embodiment, the polymer has a molecular weight of about
250 to about 7000 daltons.
[0105] "Y" is a polycationic moiety. The term polycationic moiety
refers to a compound, derivative, or functional group having a
positive charge, preferably at least 2 positive charges at a
selected pH, preferably physiological pH. Suitable polycationic
moieties include basic amino acids and their derivatives such as
arginine, asparagine, glutamine, lysine and histidine; spermine;
spermidine; cationic dendrimers; polyamines; polyamine sugars; and
amino polysaccharides. The polycationic moieties can be linear,
such as linear tetralysine, branched or dendrimeric in structure.
Polycationic moieties have between about 2 to about 15 positive
charges, preferably between about 2 to about 12 positive charges,
and more preferably between about 2 to about 8 positive charges at
selected pH values. The selection of which polycationic moiety to
employ may be determined by the type of liposome application which
is desired.
[0106] The charges on the polycationic moieties can be either
distributed around the entire liposome moiety, or alternatively,
they can be a discrete concentration of charge density in one
particular area of the liposome moiety e.g., a charge spike. If the
charge density is distributed on the liposome, the charge density
can be equally distributed or unequally distributed. All variations
of charge distribution of the polycationic moiety are encompassed
by the present invention.
[0107] The lipid "A," and the nonimmunogenic polymer "W," can be
attached by various methods and preferably, by covalent attachment.
Methods known to those of skill in the art can be used for the
covalent attachment of, "A" and "W." Suitable linkages include, but
are not limited to, amide, amine, carboxyl, carbonate, carbamate,
ester and hydrazone linkages. It will be apparent to those skilled
in the art that "A" and "W" must have complementary functional
groups to effectuate the linkage. The reaction of these two groups,
one on the lipid and the other on the polymer, will provide the
desired linkage. For example, when the lipid is a diacylglycerol
and the terminal hydroxyl is activated, for instance with NHS and
DCC, to form an active ester, and is then reacted with a polymer
which contains an amino group, such as with a polyamide (see, U.S.
Pat. Nos. 6,320,017 and 6,586,559, both of which are incorporated
herein by reference), an amide bond will form between the two
groups.
[0108] In certain instances, the polycationic moiety can have a
ligand attached, such as a targeting ligand or a chelating moiety
for complexing calcium. Preferably, after the ligand is attached,
the cationic moiety maintains a positive charge. In certain
instances, the ligand that is attached has a positive charge.
Suitable ligands include; but are not limited to, a compound or
device with a reactive functional group and include lipids,
amphipathic lipids, carrier compounds, bioaffinity.compounds,
biomaterials, biopolymers, biomedical devices, analytically
detectable compounds, therapeutically active compounds, enzymes,
peptides, proteins, antibodies, immune stimulators, radiolabels,
fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides,
liposomes, virosomes, micelles, immunoglobulins, functional groups,
other targeting moieties, or toxins.
D. siRNA
[0109] The nucleic acid component of the SNALPs typically comprise
an interfering RNA (i.e., siRNA), which can be provided in several
forms including, e.g. as one or more isolated small-interfering RNA
(siRNA) duplexes, longer double-stranded RNA (dsRNA) or as siRNA or
dsRNA transcribed from a transcriptional cassette in a DNA
plasmid.
[0110] An RNA population can be used to provide long precursor
RNAs, or long precursor RNAs that have substantial or complete
identity to a selected target sequence can be used to make the
siRNA. The RNAs can be isolated from cells or tissue, synthesized,
and/or cloned according to methods well known to those of skill in
the art. The RNA can be a mixed population (obtained from cells or
tissue, transcribed from cDNA, subtrated, selected etc.), or can
represent a single target sequence. RNA can be naturally occurring,
e.g., isolated from tissue or cell samples, synthesized in vitro,
e.g., using T7 or SP6 polymerase and PCR products or a cloned cDNA;
or chemically synthesized.
[0111] To form a long dsRNA, for synthetic RNAs, the complement is
also transcribed in vitro and hybridized to form a ds RNA. Ka
naturally occuring RNA population is used, the RNA complements are
also provided (e.g., to form dsRNA for digestion by E. coli RNAse
III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA
population, or by using RNA polymerases. The precursor RNAs are
then hybridized to form double stranded RNAs for digestion. The
dsRNAs can be directlu emcapsulated in the SNALPs or can be
digested in vitro prior to encapsulation.
[0112] Alternatively, one or more DNA plasmids encoding one or more
siRNA templates are encapsulated in a nucleic acid-lipid particle.
siRNA can be transcribed as sequences that automatically fold into
duplexes with hairpin loops from DNA templates in plasmids having
RNA polymerase HI transcriptional units, for example, based on the
naturally occurring transcription units for small nuclear RNA U6 or
human RNase P RNA H1 (see, Brummelkamp, et al., Science 296:550
(2002); Donze, et al., Nucleic Acids Res. 30:e46 (2002); Paddison,
et al., Genes Dev. 16:948 (2002); Yu, et al., Proc. Natl. Acad.
Sci. 99:6047 (2002); Lee, et al., Nat. Biotech. 20:500 (2002);
Miyagishi, et al., Nat. Biotech. 20:497 (2002); Paul, et al., Nat.
Biotech. 20:505 (2002); and Sui, et al., Proc. Natl. Acad. Sci.
99:5515 (2002)). Typically, a transcriptional unit or cassette will
contain an RNA transcript promoter sequence, such as an H1-RNA or a
U6 promoter, operably linked to a template for transcription of a
desired siRNA sequence and a termination sequence, comprised of 2-3
uridine residues and a polythymidine (T5) sequence (polyadenylation
signal) (Brummelkamp, Science, supra). The selected promoter can
provide for constitutive or inducible transcription. Compositions
and methods for DNA-directed transcription of RNA interference
molecules is described in detail in U.S. Pat. No. 6,573,099,
incorporated herein by reference. Preferably, the synthesized or
transcribed siRNA have 3' overhangs of about 1-4 nucleotides,
preferably of about 2-3 nucleotides and 5' phosphate termini
(Elbashir, et al., Genes Dev. 15:188 (2001); Nykanen, et al., Cell
107:309 (2001)). The transcriptional unit is incorporated into a
plasmid or DNA vector from which the interfering RNA is
transcribed. Plasmids suitable for in vivo delivery of genetic
material for therapeutic purposes are described in detail in U.S.
Pat. Nos. 5,962,428 and 5,910,488, both of which are incorporated
herein by reference. The selected plasmid can provide for transient
or stable delivery of a target cell. It will be apparent to those
of skill in the art that plasmids originally designed to express
desired gene sequences can be modified to contain a transcriptional
unit cassette for transcription of siRNA.
[0113] Methods for isolating RNA, synthesizing RNA, hybridizing
nucleic acids, making and screening cDNA libraries, and performing
PCR are well known in the art (see, e.g., Gubler & Hoffman,
Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al.,
supra), as are PCR methods (see U.S. Pat. Nos. 4,68,3,195 and
4,683,202; PCR Protocols: A Guide to Methods and Applications
(Innis et al., eds, 1990)). Expression libraries are also well
known to those of skill in the art. Additional basic texts
disclosing the general methods of use in this invention include
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.
1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual
(1990); and Current Protocols in Molecular Biology (Ausubel et al.,
eds., 1994)).
1. Target Genes
[0114] Generally, it is desired to deliver the SNALPS to
downregulate or silence the translation (i.e., expression) of a
gene product of interest. Suitable classes of gene products
include, but are not limited to, genes associated with viral
infection and survival, genes associated with metabolic dieases and
disorders (e.g., diseases and disorders in which the liver is the
target, and liver diseases and disorders) and disorders, genes
associated with tumorigenesis and cell transformation, angiogenic
genes, immunomodulator genes, such as those associated with
inflammatory and autoimmune responses, ligand receptor genes, and
genes associated with neurodegenerative disorders.
[0115] Genes associated with viral infection and survival include
those expressed by a virus in order to bind, enter and replicate in
a cell. Of particular interest are viral sequences associated with
chronic viral diseases. Viral sequences of particular interest
include sequences of Hepatitis viruses (Hamasaki, et al., FEBS
Lett. 543:51 (2003); Yokota, et al., EMBO Rep. 4:602 (2003);
Schlomai, et al., Hepatology 37:764 (2003); Wilson, et al., Proc.
Natl. Acad. Sci. 100:2783 (2003); Kapadia, et al., Proc. Natl.
Acad. Sci. 100:2014 (2003); and FIELDS VIROLOGY (Knipe et al. eds.
2001)), Human Immunodeficiency Virus (HIV) (Banerjea, et al., Mol.
Ther. 8:62 (2003); Song, et al., J. Virol. 77:7174 (2003);
Stephenson JAMA 289:1494 (2003); Qin, et al., Proc. Natl. Acad.
Sci. 100:183 (2003)), Herpes viruses (Jia, et al., J. Virol.
77:3301 (2003)), and Human Papilloma Viruses (HPV) (Hall, et al.,
J. Virol. 77:6066 (2003); Jiang, et al., Oncogene 21:6041 (2002)).
Examplary hepatitis viral nucleic acid sequences that can be
silenced include but are not limited to: nucleic acid sequences
involved in transcription and translation (e.g., En1, En2, X, P),
nucleic acid sequences encoding structural proteins (e.g., core
proteins including C and C-related proteins; capsid and envelope
proteins including S, M, and/or L proteins, or fragments thereof)
(see, e.g., FIELDS VIROLOGY, 2001, supra). Exemplary Hepatits C
nucleic acid sequences that can be silenced include but are not
limited to: serine proteases (e.g., NS3/NS4), helicases (e.g. NS3),
polymerases (e.g., NS5B), and envelope proteins (e.g., E1, E2, and
p7). Hepatitis A nucleic acid sequences are set forth in e.g.,
Genbank Accession No. NC.sub.--001489 ; Hepatitis B nucleic acid
sequences are set forth in, e.g., Genbank Accession No.
NC.sub.--003977; Hepatitis C nucleic acid sequences are set forth
in, e.g., Genbank Accession No. NC.sub.--004102; Hepatitis D
nucleic acid sequence are set forth in, e.g., Genbank Accession No.
NC.sub.--001653; Hepatitis E nucleic acid sequences are set forth
in e.g., Genbank Accession No. NC.sub.--001434;. and Hepatitis G
nucleic acid sequences are set forth in e.g., Genbank Accession No.
NC.sub.--001710. Silencing of sequences that encode genes
associated with viral infection and survival can conveniently be
used in combination with the administration of conventional agents
used to treat the viral condition.
[0116] Genes associated with metabolic diseases and disorders
(e.g., disorders in which the liver is the target and liver
diseases and disorders) include, for example genes expressed in,
for example, dyslipidemia (e.g., liver X receptors (e.g.,
LXR.alpha. and LXR.beta. Genback Accession No. NM.sub.--007121),
farnesoid X receptors (FXR) (Genbank Accession No.
NM.sub.--005123), sterol-regulatory element binding protein
(SREBP), Site-1 protease (SIP), 3-hydroxy-3-methylglutaryl
coenzyme-A reductase (HMG coenzyme-A reductase), Apolipoprotein
(ApoB), and Apolipoprotein (ApoE)) and diabetes (e.g., Glucose
6-phosphatase) (see, e.g., Forman et al., Cell 81:687 (1995); Seol
et al., Mol. Endocrinol. 9:72 (1995), Zavacki et al., PNAS USA
94:7909 (1997); Sakai, et al., Cell 85:1037-1046 (1996); Duncan, et
al., J. Biol. Chem. 272:12778-12785 (1997); Willy, et al., Genes
Dev. 9(9):1033-45 (1995); Lehmann, et al., J. Biol. Chem. 272
(6):3137-3140 (1997); Janowski, et al., Nature 383:728-731 (1996);
Peet, et al., Cell 93:693-704 (1998)). One of skill in the art will
appreciate that genes associated with metabolic diseases and
disorders (e.g., diseases and disorders in which the liver is a
target and liver diseases and disorders) include genes that are
expressed in the liver itself as well as and genes expressed in
other organs and tissues. Silencing of sequences that encode genes
associated with metabolic diseases and disorders can conveniently
be used in combination with the administration of conventional
agents used to treat the disease or disorder.
[0117] Examples of gene sequences associated with tumorigenesis and
cell transformation include translocation sequences such as MLL
fusion genes, BCR-ABL (Wilda, et al., Oncogene, 21:5716 (2002);
Scherr, et al., Blood 101:1566), TEL-AML1, EWS-FLI1, TLS-FUS,
PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich, et al.,
Blood 101:3157 (2003)); overexpressed sequences such as multidrug
resistance genes (Nieth, et al., FEBS Lett. 545:144 (2003); Wu, et
al, Cancer Res. 63:1515 (2003)), cyclins (Li, et al., Cancer Res.
63:3593 (2003); Zou, et al., Genes Dev. 16:2923 (2002)),
beta-Catenin (Verma, et al., Clin Cancer Res. 9:1291 (2003)),
telomerase genes (Kosciolek, et al., Mol Cancer Ther. 2:209
(2003)), c-MYC, N-MYC, BCL-2, ERBB1 and ERBB2 (Nagy, et al. Exp.
Cell Res. 285:39 (2003)); and mutated sequences such as RAS
(reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158
(2002)). Silencing of sequences that encode DNA repair enzymes fmd
use in combination with the administration of chemotherapeutic
agents (Collis, et al., Cancer Res. 63:1550 (2003)). Genes encoding
proteins associated with tumor migration are also target sequences
of interest, for example, integrins, selectins and
metalloproteinases. The foregoing examples are not exclusive. Any
whole or partial gene sequence that facilitates or promotes
tumorigenesis or cell transformation, tumor growth or tumor
migration can be included as a template sequence
[0118] Angiogenic genes are able to promote the formation of new
vessels. Of particular interest is Vascular Endothelial Growth
Factor (VEGF) (Reich, et al., Mol. Vis. 9:210 (2003)).
[0119] Immunomodulator genes are genes that modulate one or more
immune responses. Examples of immunomodulator genes include
cytokines such as growth factors (e.g., TGF-.alpha., TGF-.beta.,
EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins
(e.g., IL-2, IL-4, IL-12 (Hill, et al., J. Immunol. 171:691
(2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g.,
IFN-.alpha., IFN-.beta., IFN-.gamma., etc.) and TNF. Fas and Fas
Ligand genes are also immunomodulator target sequences of interest
(Song, et al., Nat. Med. 9:347 (2003)). Genes encoding secondary
signaling molecules in hematopoietic and lymphoid cells are also
included in the present invention, for example, Tec family kinases,
such as Bruton's tyrosine kinase (Btk) (Heinonen, et al., FEBS
Lett. 527:274 (2002)).
[0120] Cell receptor ligands include ligands that are able to bind
to cell surface receptors (e.g., insulin receptor, EPO receptor,
G-protein coupled receptors, receptors with tyrosine kinase
activity, cytokine receptors, growth factor receptors, etc.), to
modulate (e.g,. inhibit, activate, etc.) the physiological pathway
that the receptor is involved in (e.g., glucose level modulation,
blood cell development, mitogenesis, etc.). Examples of cell
receptor ligands include cytokines, growth factors, interleukins,
interferons, erythropoietin (EPO), insulin, glucagon, G-protein
coupled receptor ligands, etc.). Templates coding for an expansion
of trinucleotide repeats (e.g., CAG repeats), find use in silencing
pathogenic sequences in neurodegenerative disorders caused by the
expansion of trinucleotide repeats, such as spinobulbular muscular
atrophy and Huntington's Disease (Caplen, et al., Hum. Mol. Genet.
11:175 (2002)).
IV. Preparation of SNALPs
[0121] The present invention provides methods for preparing
serum-stable nucleic acid-lipid particles such that the nucleic
acid (e.g., siRNA or plasmid encoding siRNA) is encapsulated in a
lipid bilayer and is protected from degradation. The SNALPs made by
the methods of this invention are typically about 50 to about 150
nm in diameter. They generally have a median diameter of less than
about 150 nm, more typically a diameter of less than about 100 nm,
with a majority of the particles having a median diameter of about
65 to 85 nm. The particles can be formed by using any method known
in the art including, e.g., a detergent dialysis method or by
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 plasmid or other nucleic acid (i.e., siRNA) is
contacted with a detergent solution of cationic lipids to form a
coated nucleic acid complex. These coated nucleic acids can
aggregate and precipitate. However, the presence of a detergent
reduces this aggregation and allows the coated nucleic acids to
react with excess lipids (typically, noncationic lipids) to form
particles in which the plasmid or other nucleic acid is
encapsulated in a lipid bilayer. The methods described below for
the formation of nucleic acid-lipid particles using organic
solvents follow a similar scheme. Exemplary methods of making
SNALPS are disclosed in U.S. Pat. Nos. 5,705,385; 5,981,501;
5,976,567; 6,586,410; 6,534,484; U.S. application Ser. No.
09/553,639; U.S.P.A. Publication Nos. 2002/0072121 and
2003/0077829)WO 96/40964; and WO 00/62813.
[0122] In one embodiment, the present invention provides nucleic
acid-lipid particles produced via a process that includes providing
an aqueous solution in a first reservoir, and providing an organic
lipid solution in a second reservoir, and mixing the aqueous
solution with the organic lipid solution such that the organic
lipid solution mixes with the aqueous solution so as to
substantially instantaneously produce a liposome encapsulating the
nucleic acid. This process and the apparatus for carrying this
process is described in detail in U.S. patent application Ser. No.
10/611, 274 filed Jun. 30, 2003, which is incorporated herein by
reference.
[0123] The action of continuously introducing lipid and buffer
solutions into a mixing environment, such as in a mixing chamber,
causes a continuous dilution of the lipid solution with the buffer
solution, thereby producing a liposome substantially
instantaneously upon mixing. As used herein, the phrase
"continuously diluting a lipid solution with a buffer solution"
(and variations) generally means'that the lipid solution is diluted
sufficiently rapidly in an hydration process with sufficient force
to effectuate vesicle generation. By mixing the aqueous solution
with the organic lipid solution, the organic lipid solution
undergoes a continuous stepwise dilution in the presence of the
buffer (aqueous) solution to produce a liposome.
[0124] In some embodiments, the particles are formed using
detergent dialysis. Thus, the present invention provides a method
for the preparation of serum-stable nucleic acid-lipid particles,
comprising: [0125] (a) combining a nucleic acid with cationic
lipids in a detergent solution to form a coated nucleic acid-lipid
complex; [0126] (b) contacting noncationic lipids with the coated
nucleic acid-lipid complex to form a detergent solution comprising
a nucleic acid-lipid complex and noncationic lipids; and [0127] (c)
dialyzing the detergent solution of step (b) to provide a solution
of serum-stable nucleic acid-lipid particles, wherein the nucleic
acid is encapsulated in a lipid bilayer and the particles are
serum-stable and have a size of from about 50 to about 150 nm.
[0128] An initial solution of coated nucleic acid-lipid complexes
is formed by combining the nucleic acid with the cationic lipids in
a detergent solution.
[0129] 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.
[0130] The cationic lipids and nucleic acids 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 nucleic acid 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 200 .mu.g/mL, and more preferably from about 50
.mu.g/mL to about 100 .mu.g/mL. The combination of nucleic acids
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 nucleic acids and
cationic lipids can be combined in the detergent solution and
warmed to temperatures of up to about 37.degree. C. For nucleic
acids which are particularly sensitive to temperature, the coated
complexes can be formed at lower temperatures, typically down to
about 4.degree. C.
[0131] In a preferred embodiment, the nucleic acid to lipid ratios
(mass/mass ratios) in a formed SNALP 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 nucleic acid as well as the empty liposomes. In
another preferred embodiment, the SNALP preparation uses about 400
.mu.g nucleic acid per 10 mg total lipid or a nucleic acid 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 nucleic
acid.
[0132] The detergent solution of the coated nucleic acid-lipid
complexes is then contacted with non-cationic lipids to provide a
detergent solution of nucleic acid-lipid complexes and non-cationic
lipids. The non-cationic lipids which are useful in this step
include, diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide
or sphingomyelin. The acyl groups in these lipids are preferably
acyl groups derived from fatty acids having C.sub.10-C.sub.24
carbon chains. More preferably the acyl groups are lauroyl,
myristoyl, palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the non-cationic lipid will be
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 nucleic acid-lipid
particles will be fusogenic particles with enhanced properties in
vivo and the non-cationic lipid will be DSPC or DOPE. In addition,
the nucleic acid-lipid particles of the present invention may
further comprise cholesterol. In other preferred embodiments, the
noncationic lipids will further comprise polyethylene glycol-based
polymers such as PEG 2000, PEG 5000 and polyethylene glycol
conjugated to a diacylglycerol, a ceramide or a phospholipid, as
described in U.S. Pat. No. 5,820,873 and in co-pending patent
application Ser. No. 10/136,707 (published as U.S.P.A. Publication
No. 2003/0077829), both of which are incorporated herein by
reference. In further preferred embodiments, the noncationic lipids
will further comprise polyethylene glycol-based polymers such as
PEG 2000, PEG 5000 and polyethylene glycol conjugated to a
dialkyloxypropyl.
[0133] The amount of noncationic lipid which is used in the present
methods is typically about 2 to about 20 mg of total lipids to 50
.mu.g of nucleic acid. Preferably the amount of total lipid is from
about 5 to about 10 mg per 50 .mu.g of nucleic acid.
[0134] Following formation of the detergent solution of nucleic
acid-lipid complexes and non-cationic lipids, the detergent is
removed, preferably by dialysis. The removal of the detergent
results in the formation of a lipid-bilayer which surrounds the
nucleic acid providing serum-stable nucleic acid-lipid particles
which have a size of from about 50 nm to about 150 nm. The
particles thus formed do not aggregate and are optionally sized to
achieve a uniform particle size.
[0135] The serum-stable nucleic acid-lipid particles can be sized
by any of the methods available for sizing liposomes. The sizing
may be conducted in order to achieve a desired size range and
relatively narrow distribution of particle sizes.
[0136] 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.
[0137] Extrusion of the particles through a small-pore
polycarbonate membrane or an asymmetric ceramic membrane is also an
effective method for reducing particle sizes to a relatively
well-defined size distribution. Typically, the suspension is cycled
through the membrane one or more times until the desired particle
size distribution is achieved. The particles may be extruded
through successively smaller-pore membranes, to achieve a gradual
reduction in size.
[0138] In another group of embodiments, the present invention
provides a method for the preparation of serum-stable nucleic
acid-lipid particles, comprising: [0139] (a) preparing a mixture
comprising cationic lipids and noncationic lipids in an organic
solvent; [0140] (b) contacting an aqueous solution of nucleic acid
with said mixture in step (a) to provide a clear single phase; and
[0141] (c) removing said organic solvent to provide a suspension of
nucleic acid-lipid particles, wherein said nucleic acid is
encapsulated in a lipid bilayer, and said particles are stable in
serum and have a size of from about 50 to about 150 nm.
[0142] The nucleic acids (or plasmids), cationic lipids and
noncationic lipids which are useful in this group of embodiments
are as described for the detergent dialysis methods above.
[0143] 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
nucleic acid 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 may also be used in the present invention.
[0144] Contacting the nucleic acid with the organic solution of
cationic and non-cationic lipids is accomplished by mixing together
a first solution of nucleic acid, 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.
[0145] After the nucleic acid has been contacted with the organic
solution of lipids, the organic solvent is removed, thus forming an
aqueous suspension of serum-stable nucleic acid-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.
[0146] The serum-stable nucleic acid-lipid particles thus formed
will typically be sized from about 50 urn to 150 nm. To achieve
further size reduction or homogeneity of size in the particles,
sizing can be conducted as described above.
[0147] In other embodiments, the methods will further comprise
adding nonlipid polycations which are useful to effect the delivery
to cells using the present compositions.
[0148] 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 heaxadimethrine. Other suitable polycations include,
for example, salts of poly-L-omithine, poly-L-arginine,
poly-L-lysine, poly-D-lysine, polyallylamine and
polyethyleneimine.
[0149] In certain embodiments, the formation of the nucleic
acid-lipid particles 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.
[0150] When formation of the complexes is carried out in a
mono-phase system, the cationic lipids and nucleic acids 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 nucleic acid (which is
present in the aqueous phase), and "pull" it into the organic
phase.
[0151] In another embodiment, the present invention provides a
method for the preparation of nucleic acid-lipid particles,
comprising: [0152] (a) contacting nucleic acids with a solution
comprising noncationic lipids and a detergent to form a nucleic
acid-lipid mixture; [0153] (b) contacting cationic lipids with the
nucleic acid-lipid mixture to neutralize a portion of the negative
charge of the nucleic acids and form a charge-neutralized mixture
of nucleic acids and lipids; and [0154] (c) removing the detergent
from the charge-neutralized mixture to provide the nucleic
acid-lipid particles in which the nucleic acids are protected from
degradation.
[0155] In one group of embodiments, the solution of non-cationic
lipids and detergent is an aqueous solution. Contacting the nucleic
acids with the solution of non-cationic lipids and detergent is
typically accomplished by mixing together a first solution of
nucleic acids 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 nucleic acid
solution is also a detergent solution. The amount of non-cationic
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.
[0156] In some embodiments, the nucleic acids are precondensed as
described in, e.g., U.S. patent application Ser. No.
09/744,103.
[0157] The nucleic acid-lipid mixture thus formed is contacted with
cationic lipids to neutralize a portion of the negative charge
which is associated with the nucleic acids (or other polyanionic
materials) present. The amount of cationic lipids used will
typically be sufficient to neutralize at least 50% of the negative
charge of the nucleic acid. 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, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. These
lipids and related analogs have been described in U.S. Pat. Nos.
5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and
5,785,992, the disclosures of each of which are incorporated herein
by reference.
[0158] Contacting the cationic lipids with the nucleic acid-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 nucleic acid lipid mixture. Upon mixing
the two solutions (or contacting in any other manner), a portion of
the negative charge associated with the nucleic acid is
neutralized. Nevertheless, the nucleic acid remains in an
uncondensed state and acquires hydrophilic characteristics.
[0159] After the cationic lipids have been contacted with the
nucleic acid-lipid mixture, the detergent (or combination of
detergent and organic solvent) is removed, thus forming the nucleic
acid-lipid particles. The methods used to remove the detergent will
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.
[0160] The particles thus formed will typically be sized from about
100 nm to several microns. To achieve further size reduction or
homogeneity of size in the particles, the nucleic acid-lipid
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.
[0161] In other embodiments, the methods will further comprise
adding nonlipid polycations which are useful to effect 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.
[0162] In another aspect, the present invention provides methods
for the preparation of nucleic acid-lipid particles, comprising:
[0163] (a) contacting an amount of cationic lipids with nucleic
acids 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 nucleic acid-lipid
complex; [0164] (b)contacting the hydrophobic, nucleic acid-lipid
complex in solution with non-cationic lipids, to provide a nucleic
acid-lipid mixture; and [0165] (c)removing the organic solvents
from the nucleic acid-lipid mixture to provide nucleic acid-lipid
particles in which the nucleic acids are protected from
degradation.
[0166] The nucleic acids, non-cationic 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.
[0167] In preferred embodiments, the cationic lipids are DODAC,
DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations thereof. In other
preferred embodiments, the noncationic lipids are ESM, DOPE, DOPC,
DSPC, polyethylene glycol-based polymers (e.g., PEG 2000, PEG 5000,
PEG-modified diacylglycerols, or PEG-modified dialkyloxypropyls),
distearoylphosphatidylcholine (DSPC), cholesterol, or combinations
thereof. In still other preferred embodiments, the organic solvents
are methanol, chloroform, methylene chloride, ethanol, diethyl
ether or combinations thereof.
[0168] In one embodiment, the nucleic acid is a plasmid from which
an interfering RNA is transcribed; the cationic lipid is DODAC,
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.
[0169] As above, contacting the nucleic acids with the cationic
lipids is typically accomplished by mixing together a first
solution of nucleic acids 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
non-cationic lipids and the removal of the organic solvent. The
addition of the non-cationic lipids is typically accomplished by
simply adding a solution of the non-cationic 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.
[0170] The amount of non-cationic 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 nucleic acid-lipid
complex. Preferably, the amount is from about 0.5 to about 9 times
the amount of cationic lipids used.
[0171] In yet another aspect, the present'invention provides
nucleic acid-lipid particles which are prepared by the methods
described above. In these embodiments, the nucleic acid-lipid
particles are either net charge neutral or carry an overall charge
which provides the particles with greater gene lipofection
activity. Preferably, the nucleic acid component of the particles
is a nucleic acid which interferes with the production of an
undesired protein. In a preferred embodiment, the nucleic acid
comprises an interfering RNA, the noncationic lipid is egg
sphingomyelin and the cationic lipid is DODAC. In a preferred
embodiment, the nucleic acid comprises an interfering RNA, the
noncationic lipid is a mixture of DSPC and cholesterol, and the
cationic lipid is DOTMA. In other preferred embodiments, the
noncationic lipid may further comprise cholesterol.
[0172] A variety of general methods for making SNALP-CPLs
(CPL-containing SNALPs) are discussed herein. Two general
techniques include "post-insertion" technique, that is, insertion
of a CPL into for example, a pre-formed SNALP, and the "standard"
technique, wherein the CPL is included in the lipid mixture during
for example, the SNALP formation steps. The post-insertion
technique results, in SNALPs having CPLs mainly in the external
face of the SNALP bilayer membrane, whereas standard techniques
provide SNALPs having CPLs on both internal and external faces. The
method is especially useful for vesicles made from phospholipids
(which can contain cholesterol) and also for vesicles containing
PEG-lipids (such as PEG-DAGs). Methods of making SNALP-CPL, are
taught, for example, in U.S. Pat. Nos. 5,705,385, 6,586,410,
5,981,501 and 6,534,484; in U.S. application Ser. Nos. 09/553,639
and 09/839,707 (published as U.S.P.A. Publication No.
2002/0072121), as well as in PCT International Application
PCT/CA00/00451 (published as WO 00/62813), each of which is
incorporated herein by reference.
[0173] The nucleic acid-lipid particles 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.
[0174] The pharmaceutical carrier is generally added following
particle formation. Thus, after the particle is formed, the
particle can be diluted into pharmaceutically acceptable carriers
such as normal saline.
[0175] The concentration of particles in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.05%,
usually at or at least about 2.5% 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 may be increased to lower the fluid
load associated with treatment. This may be particularly desirable
in patients having atherosclerosis associated congestive heart
failure or severe hypertension. Alternatively, particles composed
of irritating lipids may be diluted to low concentrations to lessen
inflammation at the site of administration.
[0176] As described above, in some embodiments of the present
invention the nucleic acid-lipid particles comprise DAG-PEG
conjugates. In other embodiments, of the invention, the nucleic
acid-lipid particles comprise PEG-dialkyloxypropyl conjugates. It
is often desirable to include other components that act in a manner
similar to the DAG-PEG conjugates or PEG-dialkyloxypropyl
conjugates and that serve to prevent particle aggregation and to
provide a means for increasing circulation lifetime and increasing
the delivery of the nucleic acid-lipid particles 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%.
[0177] The pharmaceutical compositions of the present invention may
be sterilized by conventional, well known sterilization techniques.
Aqueous solutions can be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with a sterile aqueous solution prior to administration.
The compositions can contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, and calcium chloride.
Additionally, the particle suspension may include lipid-protective
agents which protect lipids against free-radical and
lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as alphatocopherol and water-soluble iron-specific
chelators, such as ferrioxamine, are suitable.
V. Administration of SNALPs
[0178] The SNALPs of the present invention can conveniently be used
to introduce nucleic acids into cells (e.g., to treat or prevent a
disease or disorder associated with expression of a target gene).
Accordingly, the present invention also provides methods for
introducing a nucleic acid (e.g., an interfering RNA) into a cell.
The methods are carried out in vitro or in vivo by first forming
the particles as described above, then contacting the particles
with the cells for a period of time sufficient for delivery of
siRNA to occur.
[0179] The SNALPs of the present invention can be adsorbed to
almost any cell type with which they are mixed or contacted. Once
adsorbed, the particles can either be endocytosed by a portion of
the cells, exchange lipids with cell membranes, or fuse with the
cells. Transfer or incorporation of the nucleic acid portion of the
particle can take place via any one of these pathways. In
particular, when fusion takes place, the particle membrane is
integrated into the cell membrane and the contents of the particle
combine with the intracellular fluid.
[0180] Among the cell types most often targeted for intracellular
delivery of a siRNA are neoplastic cells (e.g. tumor cells) and
hepatocytes. Other cells that can be targeted, e.g., hematopoietic
precursor (stem) cells, fibroblasts, keratinocytes, hepatocytes,
endothelial cells, skeletal and smooth muscle cells, osteoblasts,
neurons, quiescent lymphocytes, terminally differentiated cells,
slow or noncycling primary cells, parenchymal cells, lymphoid
cells, epithelial cells, bone cells, and the like. In a preferred
embodiment, hepatocytes are targeted.
[0181] To the extent that tissue culture of cells may be required,
it is well known in the art. Freshney (1994) (Culture of Animal
Cells, a Manual of Basic Technique, third edition Wiley-Liss, New
York), Kuchler et al. (1977) Biochemical Methods in Cell Culture
and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc.,
and the references cited therein provides a general guide to the
culture of cells. Cultured cell systems often will be in the form
of monolayers of cells, although cell suspensions are also
used.
A. In Vitro Gene Transfer
[0182] For in vitro applications, the delivery of siRNA can be to
any cell grown in culture, whether of plant or animal origin,
vertebrate or invertebrate, and of any tissue or type. In preferred
embodiments, the cells will be animal cells, more preferably
mammalian cells, and most preferably human cells.
[0183] Contact between the cells and the lipid nucleic acid
particles, when carried out in vitro, takes place in a biologically
compatible medium. The concentration of particles varies widely
depending on the particular application, but is generally between
about 1 .mu.mol and about 10 mmol. Treatment of the cells with the
nucleic acid-lipid particles is generally carried out at
physiological temperatures (about 37.degree. C.) for periods of
time of from about 1 to 48 hours, preferably of from about 2 to 4
hours.
[0184] In one group of preferred embodiments, a lipid nucleic acid
particle suspension is added to 60-80% confluent plated cells
having a cell density of from about 10.sup.3 to about 10.sup.5
cells/mL, more preferably about 2.times.10.sup.4 cells/mL. The
concentration of the suspension added to the cells is preferably of
from about 0.01 to 0.2 .mu.g/mL, more preferably about 0.1
.mu.g/mL.
[0185] Using an Endosomal Release Parameter (ERP) assay, the
delivery efficiency of the SNALP or other lipid-based carrier
system can be optimized. An ERP assay is described in detail in
U.S. patent application Ser. No. 10/136,707 (published as U.S.P.A.
Publication No. 2003/0077829), incorporated herein by reference.
More particularly, the purpose of an ERP assay is to distinguish
the effect of various cationic lipids and helper lipid components
of SNALPs based on their relative effect on binding/uptake or
fusion with/destabilization of the endosomal membrane. This assay
allows one to determine quantitatively how each component of the
SNALP or other lipid-based carrier system effects delivery
efficiency, thereby optimizing the SNALPs or other lipid-based
carrier systems. Usually, an ERP assay measures expression of a
reporter protein (e.g., luciferase, .beta.-galactosidase, green
fluorescent protein, etc.), and in some instances, a SNALP
formulation optimized for an expression plasmid will also be
appropriate for encapsulating an interfering RNA. In other
instances, an ERP assay can be adapted to measure downregulation of
translation of a target sequence in the presence or absence of an
interfering RNA. By comparing the ERPs for each of the various
SNALPs or other lipid-based formulations, one can readily determine
the optimized system, e.g., the SNALP or other lipid-based
formulation that has the greatest uptake in the cell.
[0186] 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 Green.TM.;
rhodamine and derivatives, such Texas red, tetrarhodimine
isothiocynate (TRITC), etc., digoxigenin, biotin, phycoerythrin,
AMCA, CyDyes.TM., and the like; radiolabels, such as .sup.3H,
.sup.125I, .sup.35S, .sup.14C, .sup.32P, .sup.33P, etc.; enzymes,
such as horse radish peroxidase, alkaline phosphatase, etc.;
spectral colorimetric labels, such as colloidal gold or colored
glass or plastic beads, such as polystyrene, polypropylene, latex,
etc. The label can be coupled directly or indirectly to a component
of the SNALP or other lipid-based carrier system 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 SNALP component, stability
requirements, and available instrumentation and disposal
provisions.
B. In Vivo Gene Transfer
[0187] In some embodiments, the SNALPs can be used for in vivo
delivery of siRNA to a wide variety of vertebrates, including
mammals such as canines, felines, equines, bovines, ovines,
caprines, rodents, lagomorphs, swines, primates, including, e.g.,
humans. In vivo delivery of the SNALPs may be local (i.e., directly
to the site of interest) or systemic (i.e., distal from the site of
interest).
[0188] Systemic delivery for in vivo gene therapy, i.e., delivery
of a therapeutic nucleic acid to a distal target cell via body
systems such as the circulation, has been achieved using nucleic
acid-lipid particles such as those disclosed in published PCT
Patent Application WO 96/40964, U.S. Pat. Nos. 5,705,385,
5,976,567, 5,981,501, and 6,410,328, each of which are incorporated
herein by reference. This, latter format provides a fully
encapsulated nucleic acid-lipid particle that protects the nucleic
acid from nuclease degradation in serum, is nonimmunogenic, is
small in size and is suitable for repeat dosing.
[0189] The SNALPs of the present invention can be administered via
any route known in the art including, e.g., intravenously,
intramuscularly, subcutaneously, intradermally, intraperitoneally,
orally, intranasally, or topically. For example, Zhu, et al.,
Science 261:209 (1993) describes the intravenous delivery of
plasmid-cationic lipid complexes; Hyde, et al., Nature 362:250
(1993) describes intranasal delivery of plasmid-liposome complexes
(i.e., lipoplexes); and Brigham, et al., Am. J. Med. Sci. 298:278
(1989), describes intravenous and intratracheal delivery of
plasmid-cationic lipid complexes. The SNALPs may be administered
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.
[0190] When preparing pharmaceutical preparations of the nucleic
acid-lipid particles of the invention, it is preferable to use
quantities of the nucleic acid-lipid particles which have been
purified to reduce or eliminate empty lipid particles or particles
with nucleic acid portion associated with the external surface. The
pharmaceutical carrier is generally added following particle
formation. Thus, after the particle is formed, the particle can be
diluted into pharmaceutically acceptable carriers.
[0191] The concentration of particles in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.05%,
usually at or at least about 2.5% 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.
[0192] The amount of particles administered will depend upon the
ratio of nucleic acid to lipid; the particular nucleic acid used,
the disease state being diagnosed; the age, weight, and condition
of the patient and the judgment of the clinician; but will
generally be between about 0.01 and about 50 mg per kilogram of
body weight; preferably between about 0.1 and about 5 mg/kg of body
weight or about 10.sup.8-10.sup.10 particles per injection.
1. Injectable Delivery
[0193] In certain circumstances it will be desirable to deliver the
SNALPs disclosed herein parenterally, intravenously,
intramuscularly, subcutaneously, intradermally, or
intraperitoneally as described in U.S. Pat. No. 5,543,158; U.S.
Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363. The SNALPs may be
locally injected to the site of interest (e.g., a site of disease
such as inflammation or neoplasia or to a target organ or tissue)
or systemically injected for broad distribution throughout the
organism. Solutions of the SNALPs may be prepared in water suitably
mixed with a surfactant. Dispersions may also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Typically, these preparations contain a preservative to
prevent the growth of microorganisms. Generally, when administered
intravenously, the nucleic acid-lipid particles formulations are
formulated with a suitable pharmaceutical carrier. Generally,
normal buffered saline (135-150 mM NaCl) will be employed as the
pharmaceutically acceptable carrier, but other suitable carriers
will suffice. Additional suitable carriers are described in, e.g.,
REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company,
Philadelphia, Pa., 17th ed. (1985). As used herein, "carrier"
includes any and all solvents, dispersion media, vehicles,
coatings, diluents, antibacterial and antifungal agents, isotonic
and absorption delaying agents, buffers, carrier solutions,
suspensions, colloids, and the like. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human. The preparation of an
aqueous composition that contains a protein as an active ingredient
is well understood in the art. Typically, such compositions are
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection can also be prepared. The preparation can also
be emulsified.
[0194] These compositions can be sterilized by conventional
liposomal sterilization techniques, such as filtration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH adjusting and buffering agents, tonicity adjusting
agents, wetting agents and the like. These compositions can be
sterilized using the techniques referred to above or,
alternatively, they can be produced under sterile conditions. The
resulting aqueous solutions may be packaged for use or filtered
under aseptic conditions and lyophilized, the lyophilized
preparation being combined with a sterile aqueous solution prior to
administration.
2. Oral Delivery
[0195] In certain applications, the SNALPs disclosed herein may be
delivered via oral administration to the individual. The active
compounds may even be incorporated with excipients and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, mouthwash, suspensions, oral sprays, syrups, wafers, and
the like (Mathiowitz et al., 1997; Hwang et al, 1998; U.S. Pat. No.
5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).
The tablets, troches, pills, capsules and thd like may also contain
the following: binders, gelatin; excipients, lubricants, or
flavoring agents. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. Of course,
any material used in preparing any dosage unit form should be
pharmaceutically pure and substantially non-toxic in the amounts
employed.
[0196] Typically, these formulations may contain at least about
0.1% of the active compound or more, although the percentage of the
active ingredient(s) may, of course, be varied and may conveniently
be between about 1 or 2% and about 60% or 70% or more of the weight
or volume of the total formulation. Naturally, the amount of active
compound(s) in each, therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in
any given unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
3. Nasal Delivery
[0197] In certain embodiments, the pharmaceutical compositions may
be delivered by intranasal sprays, inhalation, and/or other aerosol
delivery vehicles. Methods for delivering nucleic acid compositions
directly to the lungs via nasal aerosol sprays has been described
e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.
Likewise, the delivery of drugs using intranasal microparticle
resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol
compounds (U.S. Pat. No. 5,725,871) are also well-known in the
pharmaceutical arts. Likewise, transmucosal drug delivery in the
form of a polytetrafluoroetheylene support matrix is described in
U.S. Pat. No. 5,780,045.
4. Topical Delivery
[0198] In another example of their use, nucleic acid-lipid
particles 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 nucleic
acid-lipid particles can be formulated and administered as topical
creams, pastes, ointments, gels, lotions and the like.
C. Prophylactic And Therapeutic Treatment
[0199] In some embodiments, theSNALPs can be used for prophylactic
or therapeutic treatment of subjects (e.g., mammalian subjects)
with a disease or disorder associated with expression or
overexpression of a target sequence. The SNALPs are administered to
the subject in an amount sufficient to elicit a therapeutic
response in the patient. An amount adequate to accomplish this is
defined as "therapeutically effective dose or amount" or "effective
dose or amount." In determining the effective amount of the SNALP
to be administered in the treatment or prophylaxis of conditions
owing to expression or overexpression of the target gene, the
physician evaluates circulating plasma levels of the SNALPs, SNALP
toxicities, and progression of the disease associated with
expression or overexpression of the target gene. Administration can
be accomplished via single or divided doses.
[0200] For example, the SNALP can be administered to a subject
infected with, or at risk of being infected with a pathogenic
microorganism. The siRNA should preferably correspond to a sequence
that plays an essential role in the lifecycle of the microorganism,
and should also be unique to the microorganism (or at least absent
from the genome of the natural genome of a patient undergoing
therapy). The nucleic acid-lipid complex is introduced into target
cells, tissue, or organs, either ex vivo or by intravenous
injection in a therapeutically effective dose. Silencing of
sequences that encode genes associated with pathogenic infection
can conveniently be used in combination with the administration of
conventional agents used to treat the pathogenic condition. The
treatment can be administered prophylactically to persons at risk
of being infected with the pathogenic microorganism or to persons
already infected with the pathogenic microorganism.
[0201] In a preferred embodiment, the compositions and methods of
the invention can conveniently be used to treat liver disorders
such as, for example, hepatitis. For example, suitable sites for
inhibition on the Hepatitis B virus include nucleic acids sequences
encoding S, C, P, and X proteins, PRE, EnI, and EnII (see, e.g.,
FIELDS VIROLOGY, 2001, supra).) One of skill in the art will
appreciate that silencing of genes associated with hepatitis
infection can be combined with conventional treatments for
hepatitis such as, for example, immune globulin, interferon (e.g.,
pegylated and unpegylated interferon .alpha.) (see, e.g., Medina et
al., Antiviral Res. 60 (2):135-143 (2003); ribavirin (see, e.g.,
Hugle and Cerny, Rev. Med. Virol. 13 (6):361-71 (2003); adefovir
and lamivudine (see, e.g., Kock et al., Hepatology 38 (6):1410-8
(2003); prenylation inhibitors (see, e.g., Bordier et al., J. Clin.
Invest. 112 (3): 407-414 (2003)); famciclovir (see, e.g., Yurdaydin
et al., J. Hepatol. 37 (2):266-71 (2002); and saikosaponins c and d
(see, e.g., Chiang et al., Planta Med. 69 (8):705-9 (2003).
[0202] In another exemplary embodiment, the pathogenic
microorganism is HIV. For example, suitable sites for inhibition on
the HIV virus include TAR, REV or nef (Chatterjee et al., Science
258:1485-1488 (1992)). Rev is a regulatory RNA binding protein that
facilitates the export of unspliced HIV pre-mRNA from the nucleus.
Malim et al., Nature 338:254 (1989). Tat is thought to be a
transcriptional activator that functions by binding a recognition
sequence in 5' flanking mRNA. Kam & Graeble, Trends Genet.
8:365 (1992). The nucleic acid-lipid complex is introduced into
leukocytes or hemopoietic stem cells, either ex vivo or by
intravenous injection in a therapeutically effective dose. The
treatment can be administered prophylactically to persons at risk
of being infected with HIV, or to persons already infected with
HIV. Analogous methods are used for suppressing expression of
endogenous recipient cell genes encoding adhesion proteins.
[0203] In another embodiment, the compositions and methods of the
invention can conveniently be used to treat diseases and disorders
characterized by expression or overexpression of a gene or group of
genes. In some aspects, the compositions and methods of the
invention can be used to treat metabolic diseases and disorders
(e.g., diseases and disorders in which the liver is a target and
liver diseases and disorders) such as, for example, dyslipidemia
and diabetes. One of skill in the art will appreciate that
silencing of genes associated with metabolic diseases and disorders
can be combined with conventional treatments for these disorders.
For example, silencing of genes involved in dyslipidemia can be
combined treatment with, for example, statins, bile acid
sequestrants/resins and cholesterol absorption inhibitors such as
ezetimibe, plant stanols/sterols, polyphenols, as well as
nutraceuticals such as oat bran, psyllium and soy proteins,
phytostanol analogues, squalene synthase inhibitors, bile acid
transport inhibitors SREBP cleavage-activating protein (SCAP)
activating ligands, nicotinic acid (niacin), acipimox, high-dose
fish oils, antioxidants and policosanol, microsomal triglyceride
transfer protein (MTP) inhibitors, acylcoenzyme A: cholesterol
acyltransferase (ACAT) inhibitors, gemcabene, lifibrol, pantothenic
acid analogues, nicotinic acid-receptor agonists, anti-inflammatory
agents (such as Lp-PLA(2) antagonists and AGI1067) functional oils,
PPAR-.alpha., -.gamma., -.delta. agonists, as well as dual
PPAR-.alpha.,/.gamma. and `pan` PPAR-.alpha./.gamma.,/.epsilon.
agonists, cholesteryl ester transfer protein (CETP) inhibitors
(such as torcetrapib), CETP vaccines, upregulators of ATP-binding
cassette transporter (ABC) A1, lecithin cholesterol acyltransferase
(LCAT) and scavenger receptor class B Type 1 (SRB 1), as well as
synthetic apolipoprotein (Apo)E-related peptides, extended-release
niacin/lovastatin, atorvastatin/amlodipine, ezetimibe/simvastatin,
atorvastatin/CETP inhibitor, statin/PPAR agonist, extended-release
niacin/simvastatin and pravastatin/aspirin are under development,
and anti-obesity agents. (see, e.g., Bays and Stein, Expert Opin.
Pharmacother. 4 (11):1901-38 (2003)). Likewise, silencing of genes
involved in diabetes can be combined with treatment with insulin as
well as diet modification and exercise.
[0204] Analogous methods are used for suppressing expression of
endogenous recipient cell genes associated with tumorigenesis and
cell transformation, tumor growth, and tumor migration; angiogenic
genes; immunomodulator genes, such as those associated with
inflammatory and autoimmune responses; ligand receptor genes; genes
associated with neurodegenerative disorders; and additional genes
associated with viral infection and survival. Target gene sequences
of particular interest are described supra.
D. Detection of SNALPs
[0205] In some embodiments, the nucleic acid-lipid particles are
detectable in the plasma and/or cells of the subject 8, 12, 24, 36,
48,, 60, 72, 84, or 96 hours after administration of the particles.
The presence of the particles can be detected by any means known in
the art including, for example, direct detection of the particles,
detection of the interfering RNA sequence, detection of the target
sequence of interest (i.e., by detecting expression or reduced
expression of the sequence of interest), or a combination
thereof.
1. Detection of Particles
[0206] Nucleic acid-lipid particles are detected herein using any
methods known in the art. For example, a label can be coupled
directly or indirectly to a component of the SNALP or other
lipid-based carrier system using methods well known in the art. A
wide variety of labels can be used, with the choice of label
depending on sensitivity required, ease of conjugation with the
SNALP component, stability requirements, and available
instrumentation and disposal provisions. Suitable labels include,
but are not limited to, spectral labels, such as fluorescent dyes
(e.g., fluorescein and derivatives, such as fluorescein
isothiocyanate (FITC) and Oregon Green.TM.; rhodamine and
derivatives, such Texas red, tetrarhodimine isothiocynate (TRITC),
etc., digoxigenin, biotin, phycoerythrin, AMCA, CyDyes.TM., and the
like; radiolabels, such as .sup.3H, .sup.125I, .sup.35S, .sup.14C,
.sup.32P, .sup.33P, etc.; enzymes, such as horse radish peroxidase,
alkaline phosphatase, etc.; spectral colorimetric labels, such as
colloidal gold or colored glass or plastic beads, such as
polystyrene, polypropylene, latex, etc. The label can be detected
using any means, known in the art.
2. Detection of Nucleic Acids
[0207] Nucleic acids are detected and quantified herein by any of a
number of means well known to those of skill in the art. The
detection of nucleic acids proceeds by well known methods such as
Southern analysis, northern analysis, gel electrophoresis, PCR,
radiolabeling, scintillation counting, and affinity chromatography.
Additional analytic biochemical methods such as spectrophotometry,
radiography, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, may also be employed
[0208] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known
to those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in "Nucleic Acid
Hybridization, A Practical Approach," Ed. Hames, B. D. and Higgins,
S. J., IRL Press, 1985.
[0209] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system which multiplies
the target nucleic acid being detected. In vitro amplification
techniques suitable for amplifying sequences for use as molecular
probes or for generating nucleic acid fragments for subsequent
subcloning are known. Examples of techniques sufficient to direct
persons of skill through such in vitro amplification methods,
including the polymerase chain reaction (PCR) the ligase chain
reaction (LCR), Q.beta.-replicase amplification and other RNA
polymerase mediated techniques (e.g., NASBA.TM.) are found in
Sambrook, et al., In Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, 2000, and Ausubel et al., SHORT
PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (2002), as well as Mullis et al. (1987), U.S.
Pat. No. 4,683,202; PCR Protocols A Guide to Methods and
Applications (Innis et al. eds) Academic Press Inc. San Diego,
Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990),
C&EN 36; The Journal Of NIH Research, 3:81 (1991); (Kwoh et
al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989); Guatelli et al.,
Proc. Natl. Acad. Sci. USA; 87:1874 (1990); Lomell et al., J. Clin.
Chem., 35:1826 (1989); Landegren et al., Science, 241:1077 (1988);
Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560
(1989); Barringer et al., Gene, 89:117 (1990), and Sooknanan and
Malek, Biotechnology, 13:563 (1995). Improved methods of cloning in
vitro amplified nucleic acids are described in Wallace et al., U.S.
Pat. No. 5,426,039. Other methods described in the art are the
nucleic acid sequence based amplification (NASBA.TM., Cangene,
Mississauga, Ontario) and Q Beta Replicase systems.
[0210] Oligonucleotides for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as inhibitor
components are typically synthesized chemically according to the
solid phase phosphoramidite triester method described by Beaucage
and Caruthers, Tetrahedron Letts., 22 (20):1859 1862 (1981), e.g.,
using an automated synthesizer, as described in Needham VanDevanter
et al., Nucleic Acids Res., 12:6159 (1984). Purification of
oligonucleotides, where necessary, is typically performed by either
native acrylamide gel electrophoresis or by anion exchange HPLC as
described in Pearson and Regnier, J. Chrom., 255:137 149 (1983).
The sequence of the synthetic oligonucleotides can be verified
using the chemical degradation method of Maxam and Gilbert (1980)
in Grossman and Moldave (eds.) Academic Press, New York, Methods in
Enzymology, 65:499.
[0211] An alternative means for determining the level of
translation is in situ hybridization.
[0212] In situ hybridization assays are well known and are
generally described in Angerer et al., Methods Enzymol., 152:649
(1987). In an in situ hybridization assay cells are fixed to a
solid support, typically a glass slide. If DNA is to be probed, the
cells are denatured with heat or alkali. The cells are then
contacted with a hybridization solution at a moderate temperature
to permit annealing of specific probes that are labeled. The probes
are preferably labeled with radioisotopes or fluorescent
reporters.
VI. Kits
[0213] The present invention also provides nucleic acid-lipid
particles in kit form. The kit will typically be comprised of a
container which is compartmentalized for holding the various
elements of the nucleic acid-lipid particles and the endosomal
membrane destabilizer (e.g., calcium ions). The kit will contain
the compositions of the present inventions, preferably in
dehydrated form, with instructions for their rehydration and
administration. In still other embodiments, the particles and/or
compositions comprising the particles will have a targeting moiety
attached to the surface of the particle. Methods of attaching
targeting moieties (e.g., antibodies, proteins) to lipids (such as
those used in the present particles) are known to those of skill in
the art.
EXAMPLES
[0214] The following examples are offered to illustrate, but not to
limit the claimed invention. Those of skill in the art will readily
recognize a variety of noncritical parameters which can be changed
or modified to yield essentially the same results.
Example 1
SNALP Formulations Encapsulating siRNA
[0215] This example demonstrates encapsulating siRNA in SNALP
formulated with either short- or long-chain PEG-DAG and produced by
continuously mixing organic lipid and aqueous buffer solutions.
PEG-DAG lipids employed were PEG-dimyristylglycerol (C.sub.14)
(PEG-DMG) and PEG-distearylglycerol (C.sub.18) (PEG-DSG).
Anti-.beta.-galactosidase (.beta.-gal) siRNA encapsulated in
DSPC:Cholesterol:DODMA:PEG-DMG/PEG-DSG SNALP by this method
resulted in .gtoreq.90% encapsulation (Ribogreen Assay) and
.about.120 nm particle size (Malvern sizer). The preparations had
the following characteristics: [0216] 4 ml prep: anti-B-gal siRNA
in DSPC:Chol:DODMA:PEG-DMG liposomes [0217] Initial mix=94%
encapsulation [0218] Post dilution mix=98% encapsulation [0219]
Post incubation mix=97% encapsulation [0220] Post overnight
dialysis=96% encapsulation [0221] Particle size=109.7 nm [0222]
Polydispersity=0.14 [0223] 8 ml prep: anti-B-gal siRNA in
DSPC:Chol:DODMA:PEG-DMG liposomes [0224] Post dilution &
incubated mix=89% [0225] Post overnight dialysis=91% [0226]
Particle size=127.5 nm [0227] Polydispersity=0.11 [0228] 8 ml prep:
anti-B-gal siRNA in DSPC: Chol:DODMA:PEG-DSG liposomes [0229] Post
dilution & incubated mix=90% [0230] Post overnight dialysis=90%
[0231] Post sterile-filter=90% [0232] Particle size=111.6 nm [0233]
Polydispersity=0.24
Example 2
Downregulation of Intracellular Expression In Cells By Delivering
In Vitro An SNALP Formulation Encapsulating siRNA
[0234] This example demonstrates downregulation of .beta.-Gal
expression in CT26.CL25 cells delivered in vitro
DSPC:Cholesterol:DODMA:PEG-DMG liposomes encapsulating anti-(3-Gal
siRNA. The results are depicted in FIG. 1.
[0235] In vitro delivery of 0.2 .mu.g Oligofectamine-encapsulated
anti-.beta.-Gal siRNA decreased .beta.-Gal activity by about 60% in
comparison to unexposed control cells. Encapsulating 1.5 .mu.g
anti-.beta.-Gal siRNA in DSPC:Cholesterol:DODMA:PEG-DMG liposomes
decreased .beta.-Gal activity by about 30% in comparison to
unexposed control cells.
Example 3
Assays For Serum Stability
[0236] Lipid/therapeutic nucleic acid particles formulated
according to the above noted techniques can be assayed for serum
stability by a variety of methods.
[0237] For instance, in a typical DNase 1 digestion, 1 .mu.g of DNA
encapsulated in the particle of interest is incubated in a total
volume of 100 .mu.L of 5 mM HEPES, 150 mM NaCl, 10.0 mM MgCl.sub.2
pH 7.4. DNase treated samples are treated with either 100 or 10 U
of DNase I (Gibco--BRL). 1.0% Triton X-100 can be added in control
experiments to ensure that lipid formulations are not directly
inactivating the enzyme. Samples are incubated at 37.degree. C. for
30 min after which time the DNA is isolated by addition of 500
.mu.L of DNAZOL followed by 1.0 mL of ethanol. The samples are
centrifuged for 30 min at 15,000 rpm in a tabletop microfuge. The
supernatant is decanted and the resulting DNA pellet is washed
twice with 80% ethanol and dried. This DNA is resuspended in 30
.mu.L of TE buffer. 20 .mu.L of this sample is loaded on a 1.0%
agarose gel and subjected to electrophoresis in TAB buffer.
[0238] In a typical serum assay, 50 gg of DNA in free,
encapsulated, or encapsulated +0.5% Triton X100 was aliquoted into
1.5 mL Eppendorf tubes. To the tubes were added 45 .mu.l normal
murine or human serum, dH2O (to make final volume 50 .mu.L). The
tubes were sealed with parafilm and incubated at 37.degree. C. A
sample of the free, encapsulated, or encapsulated+0.5% Triton X100
not digested by nuclease (standard) was frozen in liquid nitrogen
in an Eppendorf tube and stored at -20.degree. C. Aliquots were
taken at various time points, added to GDP buffer containing
proteinase K (133 .mu.g/mL) and immediately frozen in liquid
nitrogen to stop the reaction. Once all of the time points were
collected, the samples were incubated at 55.degree. C. in a
waterbath to activate proteinase K enabling it to denature any
remaining exonuclease. Proteinase K digested samples were applied
to polyacrylamide gels to assess levels of exonuclease
degradation.
[0239] Particles disclosed above demonstrate serum stability by
showing less than 5% and preferably undetectable amounts of DNA
degradation (partial or total) as a result of such treatment, even
in the presence of 100 U DNase 1. This compares favorably to free
DNA, which is completely degraded, and plasmid/lipid complexes
(such as DOTMA or DODAC:DOPE complexes), wherein DNA is
substantially (i.e., greater than 20%, often 80%) degraded after
such treatment.
Example 4
Characterization of SNALPs
[0240] This example describes disease site targeting and gene
expression resulting from intravenous administration of SNALP in
tumor bearing mice. In this example, the encapsulated nucleic acid
is a plasmid.
[0241] The SNALP method resulted in the encapsulation of plasmid
DNA in small (diameter .about.70 nm) "stabilized nucleic acid-lipid
particles" (SNALP). SNALP consist of one plasmid per particle,
encapsulated within a lipid bilayer stabilized by the presence of a
bilayer stabilizing component, such as a poly(ethyleneglycol) (PEG)
coating. SNALP exhibited extended circulation lifetimes following
intravenous administration and promoted delivery of intact plasmid
to distal tumor sites resulting in reporter gene expression at the
disease site.
[0242] SNALP with long circulation times accumulated to levels
corresponding to five to ten percent of the total injected dose per
gram of tumor or greater than 1000 copies of plasmid DNA per cell,
giving rise to levels of gene expression that were more than two
orders of magnitude greater than those observed in any other
tissue. Interestingly, although the liver accumulated 20-30% of the
total injected dose, very low levels of gene expression were
observed in the liver. This is thought to be due to the limited
hepatocellular uptake of the PEG-ylated SNALP. See, FIGS. 7-9.
[0243] The in vivo delivery and transfection potential of nucleic
acid-lipid particles containing a bilayer stabilizing component was
further enhanced through the incorporation of a cationic PEG lipid
(CPL) consisting of a DSPE anchor, PEG.sub.3400 spacer chain and a
cationic head group. When CPL were incorporated into SNALP at
concentrations of 2 to 4 mol % the resulting CPL-SNALP were of a
similar size and stability as native SNALP. Incorporation of CPL
resulted in a dramatic increase in intracellular delivery and a
concomitant increase in transfection activity measured both in
vitro and in vivo. Specifically, CPL-SNALP yielded 10.sup.5-fold
more in vitro gene expression than native SNALP. When CPL-SNALP
were administered intravenously they yielded a substantial (250
fold) increase in hepatic gene expression compared to native SNALP.
The increase in CPL-SNALP potency was specific to the liver. The
levels of gene expression measured in the lung, kidney, spleen or
heart remained unchanged, contributing to more than two orders of
magnitude differential in the gene expression measured in the liver
vs. other organs.
[0244] These results illustrate the potential for modulating the
delivery properties of PEG-lipid containing systems while retaining
the stability and small uniform size required to achieve systemic
gene delivery. In particular they demonstrate that disease site
targeting and tissue specific gene expression can be re-programmed
by altering the lipid composition of non-viral gene delivery
systems.
Example 5
SNALPs Containing PEG-DAG Conjugates
[0245] This example demonstrates the preparation of a series of
PEG-diacylglycerol lipids (PEG-DAG) SNALPs. In this example, tlie
encapsulated nucleic acid is a plasmid.
[0246] PEG-DAG SNALP were prepared incorporating 10 mol percent
PEG-dilaurylglycerol (C.sub.12), PEG-dimyristylglycerol (C.sub.14),
PEG-dipalmitoylglycerol (C.sub.16) or PEG-disterylglycerol
(C.sub.18) and evaluated for in vitro transfection activity,
pharmacokinetics and the biodistribution of gene expression
resulting from systemic administration in tumor bearing mice.
PEG-DAG lipid containing SNALP demonstrated a similar relationship
between acyl chain length and in vitro transfection activity to
those containing PEG-ceramides. Shorter acyl chain anchors
(dimyristyl (C.sub.14) and dipalmitoyl (.sub.C16)) resulted in
SNALP particles that were less stable but have higher transfection
activity in vitro than those incorporating longer acyl chain
anchors (disteryl (C.sub.18)). Evaluation of the pharmacokinetics
of PEG-DAG containing SNALP confirmed a correlation between the
stability of the PEG lipid component and the circulation lifetime
of SNALP. SNALP containing PEG-dimyristylglycerol (C.sub.14),
PEG-dipalmitoylglycerol (C.sub.16) and PEG-disterylglycerol
(C.sub.18) demonstrated circulation half-lives of 0.75, 7 and 15
hours respectively. Extended circulation lifetime in turn
correlates with an increase in tumor delivery and concomitant gene
expression.
[0247] Upon intravenous administration, PEG-disterylglycerol
(C.sub.18) containing SNALP bypass so-called `first pass` organs,
including the lung, and elicited gene expression in distal tumor
tissue. The level of reporter gene expression observed in tumors
represents a 100 to 1000-fold differential over that observed in
any other tissue. This compared well with the behavior of SNALP
containing PEG-ceramide C.sub.20. The incorporation of PEG-DAG
in
[0248] SNALP confirmed that small size, low surface charge and
extended circulation lifetimes are prerequisite to the passive
disease site targeting leading to accumulation of plasmid DNA and
gene expression in tumors following systemic administration of
non-viral transfection systems. See, FIGS. 3-9.
Materials And Methods
Materials
[0249] DOPE and DSPC were obtained from Northern Lipids (Vancouver,
BC). DODAC and the PEG-diacylglycerols were manufactured by Inex
Pharmaceuticals (Burnaby, BC). The other materials, HEPES, OGP and
.sup.3H-cholesteryl hexadecyl ether, were obtained from a number of
different commercial sources.
[0250] DOPE:DODAC:PEG-Diacylglycerols (82.5:7.5:10) large
unilamellar vesicles were prepared via detergent dialysis in Hepes
Buffered Saline (150 mM NaCl and 10 mM HEPES) for 48 hours. Lipid
stock solutions were prepared in ethanol and then dried down to
create a lipid film which was reconstituted in final 200 mM OGP.
LUVs were labeled with .sup.3H-cholesteryl hexadecyl ether at 1
uCi/1 mg lipid. Particle sizes were determined by nicomp analysis.
Radioactivity was determined by scintillation counting with
Picofluor20.
[0251] SNALP containing PEG-Diacyglycerols were formulated via
detergent dialysis by varying the salt concentration to maximize
the percent of DNA encapsulation. Optimal salt concentration was
chosen for the 48 hour detergent dialysis. Empty vesicles were
removed by one step sucrose centrifugation. 3.5% sucrose was used
to separate out the empty particles from the plasmid-containing
PEG-Diacylglycerol formulations except for PEG-Dimyristylglycerol
containing SNALP which used 5.0% sucrose. Empty vesicles migrated
to the top of the tube which were fractioned out and removed.
In Vitro Transfection
[0252] 5.times.10.sup.4 cells/ml were plated onto 24-well plates (1
ml). Cells were left to grow for 24 hours. 500 .mu.l of
transfection media (2.5 .mu.g/well) was added and then incubated
for stated timepoints. Transfection media was aspirated after
timepoint and then exposed to complete media for another 24 hours
at 37.degree. C. in 5.0% CO.sub.2. Complete media was removed.
Cells were washed with PBS twice and stored at -70.degree. C. until
day of experiment. Cells were lysed with 150 .mu.l of 1.times.CCLR
containing protease inhibitors. Plates were shaken for 5 minutes.
20 .mu.l of each sample were assayed in duplicate on a 96-well
luminescence plate for luciferase activity.
Pharmacokinetics, Biodistribution, And In Vivo Gene Expression
[0253] Pharmacokinetics and biodistribution were all determined by
normalizing the data to the quantity of radioactivity present.
Approximately 500 .mu.l of blood was obtained by cardiac puncture.
Red blood cells and plasma were separated by centrifugation
(4.degree. C., 3000 rpm, 10 minutes) and 100 .mu.l of plasma was
used to determine radioactive counts. Organs were harvested at
specified timepoints and homogenized in lysing matrix tubes (Fast
Prep, 2.times.15 seconds, 4.5 intensity) to assay a portion of the
mixture.
[0254] Gene expression was determined by luciferase assay. Organs
were harvested, homogenized, and kept on ice throughout the
experiment. Lysates were centrifuged (10,000 rpm, 5 minutes) and 20
.mu.l of supernatant were assayed in duplicate on a 96-well
luminescence plate for luciferase activity. The results are
depicted in FIGS. 6-9.
In Vitro Gene Silencing
[0255] Cells were transfected with SPLP comprising PEG-lipid
conjugates and containing a plasmid encoding luciferase under the
control of the CMV promoter and SNALPs containing anti-luciferase
siRNA, according to the methods described above. Gene expression
was determined by luciferase assay. The results are depicted in
FIG. 17.
Example 6
Expression of Nucleic Acids Encapsulated In SPLP Comprising
PEG-Dialkyloxvpropyl Conjugates
[0256] This examples describes experiments comparing expression of
nucleic acids encapsulated in SPLP comprising PEG-dialkyloxypropyl
conjugates. All SPLP formulations comprise a plasmid encoding
luiferase under the control of the CMV promoter (pLO55)
TABLE-US-00001 A 4 Neuro- SC PBS IV 1 48 hrs Body 2a weights, B 5
Neuro- SC SPLP PEG- IV 1 48 hrs Blood 2a DSG analyses, C 5 Neuro-
SC SPLP PEG- IV 1 48 hrs Luciferase 2a A-DSA D 5 Neuro- SC SPLP
PEG- IV 1 48 hrs activity 2a A-DPA E 5 Neuro- SC SPLP PEG- IV 1 48
hrs 2a A-DMA indicates data missing or illegible when filed
[0257] The lipids (DSPC:CHOL:DODMA:PEG-Lipid) were present in the
SPLP in the following molar ratios (20:55:15:10). The following
formulations were made: [0258] A: PBS sterile filtered, 5 mL.
[0259] B: pL055-SPLP with PEG-DSG, 2 mL at 0.50 mg/mL. [0260] C:
pL055-SPLP with PEG-A-DSA, 2 mL at 0.50 mg/mL. [0261] D: pL055-SPLP
with PEG-A-DPA, 2 mL at 0.50 mg/mL. [0262] E: pL055-SPLP with
PEG-A-DMA, 2 mL at 0.50 mg/mL.
TABLE-US-00002 [0262] # Seeding Injection Collection Group Mice
date Treatment date date A 4 Day 0 PBS Day 12 Day 14 B 5 Day 0 SPLP
PEG-DSG Day 12 Day 14 C 5 Day 0 SPLP PEG-A-DSA Day 12 Day 14 D 5
Day 0 SPLP PEG-A-DPA Day 12 Day 14 E 5 Day 0 SPLP PEG-A-DMA Day 12
Day 14
[0263] 1.5'10.sup.6 Neuro2A cells were administered to each mouse
on day 0. When the tumors were of a suitable size (200-400
mm.sup.3), mice were randomized and treated with one dose of an
SPLP formulation or PBS by intravenous (1V) injection. Dose amounts
are based on body weight measurements taken on the day of dosing.
48 hours after SPLP administration, the mice were sacrificed, their
blood was collected, and the following tissues were collected
weighed, immediately frozen and stored at -80 C until further
analysis: tumor, liver (cut in 2 halves), lungs, spleen &
heart.
[0264] Gene expression in collected tissues was determined by
assaying for enzymatic activity of expressed luciferase reporter
protein. The results are shown in FIGS. 11 and 12.
[0265] The results indicate that SPLP comprising
PEG-dialkyloxypropyls (i.e., PEG-DAA) can conveniently be used to
transfect distal tumor to substantially the same extent as SPLP
comprising PEG-diacylglycerols. Moreover, the transfection levels
seen with SPLP containing PEG-dialkyloxypropyl are similar to those
seen with SPLP containing PEG-diacylglycerols (e.g. PEG-DSG). It
was also shown that similar to the PEG-diacylglycerol system, very
little transfection occurred in non-tumor tissues. Moreover, the
SPLP comprising PEG-dialkyloxypropyls exhibit reduced toxicity
compared to other SPLP formulations.
Example 7
SNALPs Containing PEG-Dialkyloxypropyl Conjugates
[0266] This example described experiments analyzing the
biodistribution (local and systemic) and pharmacokinetics of a
series of PEG-dialkyloxypropyl lipids SNALPs (i.e., SPLP containing
encapsulated siRNA.
Local Biodistribution
[0267] To determine the local distribution of SPLP resulting from
systemic administration of anti-.beta. galactosidase siRNA
containing SNALP in Neuro-2a tumor bearing mice via fluorescent
microscopy. [0268] A: PBS [0269] B: anti-.beta.gal
siRNA-Rhodamine-PE labeled-DSPC:Chol:DODMA:PEG-A-DMA SNALP
(1:20:54:15:10)
TABLE-US-00003 [0269] Group Mice Cells Treatment Timepoint Assay A
2 Neuro2A PBS 24 hr Fluorescent Photomicroscopy B 5 Neuro2A
anti-Bgal siRNA-Rhodamine- 24 hr Fluorescent PE labeled-
Photomicroscopy DSPC:Chol:DODMA:PEG-A- DMA
[0270] 1.5'10.sup.6Neuro2A cells were administered to each mouse on
day 0. When the tumors were of a suitable size (200-400 mm.sup.3,
typically day 9-12)), mice were randomized and treated with one
dose of an SNALP formulation comprising 100 .mu.g siRNA or PBS by
intravenous (IV) injection in a total volume of 230 .mu.l. Dose
amounts are based on body weight measurements taken on the day of
dosing. 24 hours after SPLP administration, the mice were
sacrificed, their blood was collected, and the following tissues
were collected weighed, immediately frozen and stored at -80 C
until further analysis: tumor, liver (cut in 2 halves), lungs,
spleen & heart.
[0271] Local distribution of the SNALP was determined by
fluorescence microscopy. Accumulation of SNALP is seen in, e.g.,
the liver, demonstrating the SNALP comprising PEG-dialkyloxypropyls
are able to extravasate, i.e., exit the circulation and home to a
target tissue or organ.
Pharmacokinetics And Systemic Biodistribution
[0272] This example illustrates the pharmacokinetics and
biodistribution of SPLPs containing a plasmid encoding luciferase
under the control of the CMV promoter (LO55) and SNALPs containing
anti-luciferase siRNA in mice seeded subcutaneously with Neuro2A
tumors.
TABLE-US-00004 Group Mice Cells Treatment imepoint ( ) A 6 Neuro2A
[3-H]CHE-L055-DSPC:Chol:DODMA:PEG-A-DMA 0.25, 1, 4, 8, 24 B 6
Neuro2A [3-H]CHE-anti-luc siRNA-DSPC:Chol:DODMA:PEG-A-DMA 0.25, 1,
4, 8, 24 C 6 Neuro2A [3-H]CHE-L055-DSPC:Chol:DODMA:PEG-C-DMA 0.25,
1, 4, 8, 24 D 6 Neuro2A [3-H]CHE-L055-pSPLP (PEI) 0.25, 1, 4, 8, 24
E 6 Neuro2A [3-H]CHE-L055-DSPC:Chol:DODMA:PEG-DSG 0.25, 1, 4, 8, 24
indicates data missing or illegible when filed
[0273] All samples are to be provided at 0.5 mg/ml nucleic acid.
The following SPLP and SNALP formulations were prepared: [0274] A.
[.sup.3H] CHE-L055-DSPC:Chol:DODMA:PEG-A-DMA (20:55:15:10) [0275]
B. [.sup.3H] CHE-anti-luc siRNA-DSPC:Chol:DODMA:PEG-A-DMA
(20:55:15:10) [0276] C. [.sup.3H] CHE-L055
-DSPC:Chol:DODMA:PEG-C-DMA (20:55:15:10) [0277] D. [.sup.3H]
CHE-L055-pSPLP (PEI) (i.e., precondensed SPLP) [0278] E. [.sup.3H]
CHE-L055-DSPC:Chol:DODMA:PEG-DSG (20:55:15:10)
TABLE-US-00005 [0278] # Seeding Injection Collection Group Mice
date Treatment date date A 6 Day 0
[3-H]CHE-L055-DSPC:Chol:DODMA:PEG-A- Day 12 July 31 DMA B 6 Day 0
[3-H]CHE-anti-luc siRNA- Day 12 July 31 DSPC:Chol:DODMA:PEG-A-DMA C
6 Day 0 [3-H]CHE-L055-DSPC:Chol:DODMA:PEG-C- Day 13 Day 14 DMA D 6
Day 0 [3-H]CHE-L055-pSPLP (PEI) Day 13 Day 14 E 6 Day 0
[3-H]CHE-L055-DSPC:Chol:DODMA:PEG-DSG Day 14 Day 15
[0279] 30 male A/J mice (Jackson Laboratories) were seeded
subcutaneously with Neuro 2A cells at a dose of 1.5.times.10.sup.6
cells in a total volume of 50 .mu.L phosphate buffered saline on
day zero. After tumors reached appropriate size (typically on day 9
or later), 200 .mu.l(100 .mu.g nucleic acid) of the SPLP or SNALP
preparations described above, were administered intravenously.
0.25, 1, 2, 4, and 8 hours after administration of SPLP or SNALP,
mice were weighed and blood (typically 25 .mu.L) was collected by
tail nick. 24 hours after administration of SPLP or SNALP, mice
were sacrificed, blood was collected and assayed for clearance of
[.sup.3H]CHE. Organs (e.g., liver, lung, spleen, kidney, heart) and
tumors were collected and evaluated for [.sup.3H]CHE accumulation.
The results are shown in FIGS. 13-16.
[0280] For all formulations, SPLP containing PEG-DSG remained in
circulation the longest, with 50% of the injected dose remaining
after 6 h. Interestingly, there appeared to be a initial rapid
clearance of pSPLP within the first 15 minutes that was not seen
for any other formulation. After 1 h the clearance profile of the
pSPLP was quite similar to SPLP. This initial rapid clearance for
the pSPLP sample may indicate that there are actually two types of
particles present, one that clears very rapidly and one that
behaves very much like SPLP.
[0281] Anti-Luc siRNA containing vesicles (SNALP) formulated with
the C14 PEG-A-DMA showed more rapid clearance from blood than SPLP
containing the C18 PEG-DSG. However, this SNALP formulation showed
significantly slower blood clearance than SPLP formulated with the
same PEG lipid. A possible reason for this result may be that siRNA
containing particles can evade the cellular immune system more
readily than plasmid containing SPLP.
[0282] SPLP comprising PEG-C-DMA demonstrated a rapid clearance
from blood, which was substantially the same as that observed for
SPLP comprisig PEG-A-DMA. For both of these formulations, the
plasma half lives were approximately 2 h, lower than for SPLP
containing C18 PEG-lipids.
[0283] SPLP containing PEG-DSG had the highest tumor accumulation
at 10.9% inject dose per gram tissue. The two SPLP formulations
containing the C14 PEG-lipids, PEG-A-DMA and PEG-C-DMA, had much
lower tumor accumulation of 6.1% and 5.9% injected dose per gram
tissue. The SiRNA SNALP had slightly more tumor accumulation than
an SPLP sample with the same PEG-lipid at 7.3%, which also
correlates relatively well with the plasma half-life for this
SNALP. The pSPLP formulation had tumor accumulation at 7.5%, which
is lower than the comparable PEG-DSG SPLP.
[0284] Accumulation of PEG-DSG containing SPLP and pSPLP in the
heart and lungs was higher than the other SPLP and SNALP, which is
consistent with the increased circulation half lives of particles
with C18 PEG-lipids. Not surprisingly, there was an inverse
relationship between plasma half-life and accumulation in the liver
for all samples tested, while no trend was apparent for sample
accumulation in the spleen. Accumulation in the kidneys was very
low for all formulations tested, with accumulation between 1.2 and
2.4% injected dose per gram tissue.
Example 8
Silencing of Gene Expression With SNALPS
[0285] This example illustrates silencing of gene expression in
Neuro 2A tumor bearing mice after co-administration of SPLPs
containing a plasmid encoding luciferase under the control of the
CMV promoter and SNALPs containing anti-luciferase siRNA.
TABLE-US-00006 1 3 Neuro- SQ PBS/PBS 48 h IV 1 24A 4 2a L055-SPLP/
24 h PBS mix 24B 4 L055-SPLP/ anti-luc siRNA liposomes mix 48A 4
L055-SPLP/ 48 h PBS mix 48B 4 L055-SPLP/ anti-luc siRNA liposomes
mix 72A 4 L055-SPLP/ 72 h PBS mix 72B 4 L055-SPLP/ anti-luc siRNA
liposomes mix indicates data missing or illegible when filed
TABLE-US-00007 1 3 Day 0 SQ PBS/PBS 48 h Day 13 Day 15 24A 4
L055-SPLP/ 24 h Day 14 PBS mix Day 14 24B 4 L055-SPLP/ anti-luc
siRNA liposomes mix 48A 4 L055-SPLP/ 48 h Day 13 PBS mix Day 13 48B
4 L055-SPLP/ anti-luc siRNA liposomes mix 72A 4 L055-SPLP/ 72 h Day
12 PBSmix Day 12 72B 4 L055-SPLP/ anti-luc siRNA liposomes mix
indicates data missing or illegible when filed
[0286] 36 male A/J mice (Jackson Laboratories) were seeded
subcutaneously with Neuro 2A cells at a dose of 1.5.times.10.sup.6
cells in a total volume of 50 .mu.L phosphate buffered saline on
day zero. Once tumors reached appropriate size (typically on day 9
or later), 200-240 .mu.l PBS, SPLP, or SNALP formulations (100
.mu.g nucleic acid total) prepared as described in Example 6 above,
were administered intravenously. 24, 48, or 72 after administration
of PBS, SPLP or a mixture of SPLP and SNALP, mice were sacrificed
and organs (e.g., liver, lung, spleen, kidney, heart) and tumors
were collected and evaluated for luciferase activity. The results
are shown in FIGS. 18-22.
[0287] The results demonstrate that co-administration of pL055 SPLP
and anti-luc siRNA SNALP (both containing PEG-A-DMA) maximally
decreases luciferase gene expression by 40% forty-eight hours after
a single iv dose.
Example 9
Synthesis of PEG-Dialkyloxvpropyls (PEG-DAA's)
[0288] The following example illustrates the synthesis of three
PEG-lipids, PEG-A-DMA (7), PEG-C-DMA (8), and PEG-S-DMA (9). They
have a common precursor, the amine lipid
1,2-dimyristyloxypropylamine (5). This lipid has alkyl chains 14
carbon units (C.sub.14) in length. Other PEG DAAs suitable for use
in the present invention can be synthesized using similar
protocols. For instance, PEG-A-DSA and PEG-C-DSA can be synthesized
by using the C.sub.18 analogue of (5). The C.sub.18 analogue can be
synthesized by simply substituting an equimolar amount of stearyl
bromide for myristyl bromide in the very first step (synthesis of
compound (1)).
1. Preparation of 1,2-Dimyristyloxy-3-allyloxypropane (1)
##STR00005##
[0290] Benzene (250 ml) was added to 95% sodium hydride (11.4 g,
450 0 mmol), and the flask was flushed with nitrogen and sealed. A
solution of 3-allyloxy-1,2-propanediol (6.6 g, 50.0 mmol) in
benzene (75 ml) was added'to the flask. Using a syringe, 97%
1-bromotetradecane (36.7 ml, 120.0 mmol) was added to the flask and
the reaction was left to reflux overnight under a constant stream
of nitrogen. Once cooled to room temperature, the excess sodium
hydride was slowly quenched with ethanol until no further
effervescence was observed. The solution was transferred to a
separatory funnel with benzene (250 ml) and washed with distilled
water (3.times.200 ml). The organic layer was dried with magnesium
sulfate and the solvent removed on the rotary evaporator to yield a
colourless oil. TLC (5% ether-hexane, developed in Molybdate)
indicated that most of the starting material had reacted to form
product. This resulting product was further purified by flash
column chromatography (1-5% ether-hexane) to yield 15.0 g (57.3%)
of 1,2-dimyristyloxy-3-allyloxypropane 1.
2. Preparation of 1,2-Dimyristyloxypropan-3-ol (2)
##STR00006##
[0292] 1,2-Dimyristyloxy-3-allyloxypropane 1 (15.0 g, 28.6 mmol)
was dissolved in ethanol (250 ml). Trifluoroacetic acid (20 ml) was
added, followed by tetrakis(triphenylphosphine)palladiuin(0) (4.5
g, 3.9 mmol). The flask was wrapped in tin foil and flushed with
nitrogen to reduce exposure to light and air, then left to stir at
80.degree. C. overnight. The ethanol was removed on the rotary
evaporator. TLC (100% CHCl.sub.3, developed in Molybdate) indicated
that most of the starting material had reacted to form product.
This resulting product was further purified by flash column
chromatography (100% DCM) to yield 11.5 g (83.1%)
1,2-dimyristyloxypropan-3-ol 2.
3. Preparation of O-(2,3-Dimyristyloxypropyl)methanesulphonate
(3)
##STR00007##
[0294] A flask containing 97% methanesulphonic anhydride (8.4 g,
48.0 mmol) was flushed with nitrogen and dissolved in anhydrous
dichloromethane (50 ml). Anhydrous pyridine (3.9 ml, 48.0 mmol) was
slowly added, forming a white precipitate. A solution of
1,2-dimyristyloxypropan-3-ol 15 (11.5 g, 24.0 mmol) in anhydrous
dichloromethane (100 ml) was added and the reaction was left to
stir overnight at room temperature. The solution was transferred to
a separatory funnel with dichloromethane (100 ml) and was washed
with distilled water (3.times.100 ml). The combined aqueous washes
were then back-extracted with dichloromethane (100 ml). The
combined organic layers were dried with magnesium sulfate and the
dichloromethane was removed on the rotary evaporator to yield a
colourless oil. TLC (100% CHCl.sub.3, developed in Molybdate)
indicated that the starting material had all reacted to form
product. This reaction yielded 11.9 g of crude
O-(2,3-dimyristyloxypropyl)methanesulphonate 3.
4. Preparation of N-(2, 3-Dimyristyloxypropyl)phthalimide (4)
##STR00008##
[0296] Crude O-(2,3-dimyristyloxypropyl)methanesulphonate 3 (14.2
g, 25.3 mmol) and potassium phthalimide (13.9 g, 75.0 mmol) were
flushed with nitrogen and dissolved in anhydrous
N,N-dimethylformamide (250 ml). The reaction was left to stir at
70.degree. C. overnight under a constant stream of nitrogen. The
N,N-dimethylformamide was removed on the rotary evaporator using a
high vacuum pump instead of the usual aspirator. The residue was
dissolved in chloroform (300 ml) and transferred to a separatory
funnel with a chloroform rinse (50 ml), then washed with distilled
water and ethanol (3.times.300 ml distilled water, 50 ml ethanol).
The combined aqueous washes were back-extracted with chloroform
(2.times.100 ml). The combined organic layers were dried with
magnesium sulfate and the chloroform was removed on the rotary
evaporator. TLC (30% ether-hexane, developed in Molybdate)
indicated that the starting material had reacted to form product.
This reaction yielded 13.5 g of crude
N-(2,3-dimyristyloxypropyl)phthalimide 4.
5. Preparation of 1,2-Dimyristyloxypropylamine (5)
##STR00009##
[0298] Crude N-(2,3-dimyristyloxypropyl)phthalimide 4 (20.0 g, 25.0
mmol) was dissolved in ethanol (300 ml). Hydrazine monohydrate (20
ml, 412.3 mmol) was added and the reaction was left to reflux
overnight. The ethanol was removed on the rotary evaporator and the
residue was redissolved in chloroform (200 ml). The precipitate was
filtered off and the chloroform was removed on the rotary
evaporator. TLC (10% MeOH--CHCl.sub.3, developed in Molybdate)
indicated that most of the starting material had reacted to form
product. This resulting product was further purified by flash
column chromatography (0-5% MeOH--CHCl.sub.3) to yield 10.4 g
(89.7% over three steps from 1,2-dimyristyloxypropan-3-ol 2) of
1,2-dimyristyloxypropylamine 5.
6. Preparation of Methoxy PEG.sub.2000 Acetic Acid (6)
##STR00010##
[0300] A 10% solution of concentrated sulfuric acid (20 ml) in
water (180 ml) was added to sodium dichromate (3.0 g, 10 mmol).
PEG.sub.2000 methyl ether (20.0 g, 10 mmol) was dissolved in this
bright orange solution and the reaction was left to stir at room
temperature overnight.
[0301] The product was then extracted with chloroform (3.times.250
ml) leaving the dark blue colour in the aqueous layer. The
chloroform solvent was removed on the rotary evaporator, resulting
in a pale blue wax. TLC (13% MeOH--CHCl.sub.3, developed in iodine)
indicated that most of the starting material had reacted to form
product. This crude material was then further purified by flash
column chromatography (0-15% MeOH--CHCl.sub.3). The resulting
product was then crystallized in ether to yield 5.6 g (27.1%) of
methoxy PEG.sub.2000 acetic acid 6 as a white solid.
7. Preparation of N-(2, 3-dimyristyloxypropyl) Amide PEG.sub.2000
Methyl Ether (7)
##STR00011##
[0303] For preparation of N-(2,3-dimyristyloxypropyl) amide
PEG.sub.2000 methyl ether (i.e., PEG-A-DMA), methoxy PEG.sub.2000
acetic acid 6 (3.4 g, 1.7 mmol) was dissolved in benzene (40 ml)
and flushed with nitrogen. Oxalyl chloride (1.7 ml, 2.5 g, 20 mmol)
was slowly added by a syringe and needle through the subaseal. This
reaction was left to stir for 2 hours then the benzene solvent was
removed on the rotary evaporator. 2,3-myristylyloxypropylamine 5
(0.87 g, 1.8 mmol) was added to the flask, followed by anhydrous
dichloromethane (40 ml) and triethylamine (1.5 ml, 10 mmol). The
reaction was left to stir for 48 hours. Distilled water (250 ml)
was added, the solution was acidified with hydrochloric acid (1.5
ml) and shaken, and the organic layer was collected. The product
was extracted from the aqueous layer with chloroform (2.times.65
ml). The combined organic layers were dried with magnesium sulfate.
The chloroform was removed on the rotary evaporator to yield a
yellow solid. TLC (10% MeOH--CHCl.sub.3, developed in copper
sulphate and iodine) indicated that most of the starting material
had reacted to form product. This crude material was further
purified by flash column chromatography (0-7% MeOH--CHC1.sub.3). It
was then decolourized by adding activated charcoal (2 g) and
ethanol (100 ml) and allowing the mixture to rotate at 55.degree.
C. on the rotary evaporator for 30 minutes. The charcoal was
filtered off and the ethanol was removed on the rotary evaporator.
The product was lyophilized to yield 1.7 g (38.1%) of
N-(2,3-dimyristyloxypropyl)amide PEG.sub.2000 methyl ether 7 as a
fluffy white powder.
8. Preparation of N-(2,3-dimyristyloxypropyl) Carbamate
PEG.sub.2000 Methyl Ether (8)
##STR00012##
[0305] For preparation of N-(2,3-dimyristyloxypropyl) carbamate
PEG.sub.2000 methyl ether (i.e., PEG-C-DMA), steps 1-5 described
above were followed. Then PEG.sub.2000 methyl ether (2.0 g, 1.0
mmol) was flushed with nitrogen and dissolved in anhydrous
dichloromethane (15 ml). Diphosgene (300 .mu.l, 2.5 mmol) was added
and the reaction was left to stir at room temperature for 3 hours.
The dichloromethane was removed on the rotary evaporator and any
remaining diphosgene was removed using the high vacuum pump. The
flask was flushed with nitrogen and 2,3-dimyristyloxypropylamine 5
(0.7 g, 1.5 mmol) was added. This was dissolved in anhydrous
dichloromethane (15 ml), triethylamine was added (280 ul), and the
reaction was left to stir at room temperature overnight. The
solution was transferred to a separatory funnel with
dichloromethane (5 ml) and washed with distilled water (2.times.20
ml). The organic layer was dried with magnesium sulfate and the
dichloromethane was removed on the rotary evaporator. TLC (3%
MeOH--CHCl.sub.3, developed in Molybdate and iodine) showed that
most of the starting material had reacted to form product. This
resulting product was further purified by flash column
chromatography (1.5-10% MeOH--CHCl.sub.3) to yield 1.2 g (46.5%) of
N-(2,3-dimyristyloxypropyl) carbamate PEG.sub.2000 methyl ether
8.
9. Preparation of N-(2,3-dimyristyloxypropyl) Succinamide
PEG.sub.2000 Methyl Ether (13)
[0306] For preparation of N-(2,3-dimyristyloxypropyl) succinamide
PEG.sub.2000 methyl ether (13), steps 1-5 described above were
followed. The remaining procedure follows"
a. Preparation of PEG.sub.2000 Mesylate (9)
##STR00013##
[0308] Mesyl anhydride (8.2 g, 47.1 mmol) was dissolved in
anhydrous chloroform (80 ml). Pyridine (3.8 ml, 47.0 mmol) was
added to the solution and fuming was observed while a white
precipitate formed. A solution of PEG.sub.2000 methyl ether (31.5
g, 15.5 mmol) in anhydrous chloroform (70 ml) was added and the
reaction was left to stir for 3 hours. The white precipitate that
had formed was filtered off and the chloroform solvent of the
filtrate was removed on the rotary evaporator. TLC (5%
MeOH--CHCl.sub.3, developed in iodine) indicated that most of the
starting material had reacted to form product. This product was
further purified by flash column chromatography (0-10%
MeOH--CHCl.sub.3) to yield 30.1 g (92.8%) of PEG.sub.2000 mesylate
9 as a white solid.
b. Preparation of PEG.sub.2000 Phthalimide (10)
##STR00014##
[0310] Potassium phthalimide (11.1 g, 59.7 mmol) was dissolved in
anhydrous N,N-Dimethylformamide (400 ml). A solution of
PEG.sub.2000 mesylate 9 (35.0 g, 16.7 mmol) in anhydrous
N,N-Dimethylformamide (100 ml) was added to the flask and the
reaction was left to stir at 75.degree. C. overnight. The
N,N-Dimethylformamide solvent was removed on the rotary evaporator
using a high vacuum pump instead of the usual aspirator. The
resulting product was dissolved in dichloromethane (250 ml) and
washed with distilled water (2.times.250 ml) and brine (250 ml).
The dichloromethane solvent of the combined organic layers was
removed on the rotary evaporator. TLC (7% MeOH--CHCl.sub.3,
visualized with UV light and Mary's Reagent) indicated that most of
the starting material had reacted to form product. This resulting
product was further purified by flash column chromatography (0-10%
MeOH--CH.sub.2Cl.sub.2). The product was crystallized from ether to
yield 19.4 g (54.1%) of the PEG.sub.2000 phthalimide 10.
c. Preparation of PEG.sub.2000 Amine (11)
##STR00015##
[0312] PEG.sub.2000 phthalimide 10 (10.3g, 4.8 mmol) was dissolved
in ethanol (200 ml). Hydrazine monohydrate (6.0 ml, 123.7 mmol) was
slowly added and the reaction was left to reflux at 100.degree. C.
overnight. The white precipitate was filtered off and the ethanol
solvent was removed on the rotary evaporator. The resulting product
was dissolved in chloroform and the remaining white solid that was
insoluble in the chloroform was filtered off and again the
chloroform was removed on the rotary evaporator. TLC (10%
MeOH--CHCl.sub.3, developed in iodine, Molybdate and Mary's
Reagent) indicated that all the starting material had reacted to
form product. This product was then crystallized from ether to
yield 9.0 g (93.0%) of PEG.sub.2000 amine 11 as a white powder.
d. Preparation of PEG.sub.2000 Succinamide (12)
##STR00016##
[0314] PEG.sub.2000 amine 11 (9.0 g, 4.4 mmol) and succinic
anhydride (3.8 g, 38.1 mmol) were dissolved in pyridine (100 ml)
and the reaction was left to stir overnight. The pyridine solvent
was removed on the rotary evaporator at 60.degree. C. The residue
was dissolved in distilled water (100 ml), acidified with
hydrochloric acid, extracted with dichloromethane (100 ml,
2.times.70 ml), and dried with magnesium sulfate. TLC (10%
MeOH--CHCl.sub.3, developed in iodine) indicated that most of the
starting material had reacted to form product. This product was
further purified by flash column chromatography (0-10%
MeOH--CHCl.sub.3) to yield 5.2 g (55.9%) of PEG.sub.2000
succinamide 12.
e. Preparation of N-(2,3-dimyristyloxypropyl) Succinamide
PEG.sub.2000 methyl Ether (13)
##STR00017##
[0316] PEG.sub.2000 succinamide (2.0 g, 0.9 mmol) and
N-hydroxysuccinamide (0.2 g, 2.0 mmol) were dissolved in anhydrous
chloroform (10 ml). Then, a solution of
1,3-Dicyclohexyl-carbodiimide (0.3 g, 1.5 mmol) in anhydrous
chloroform (5 ml) was added, and the reaction was left to stir for
an hour. A solution of 1,2-dimyristyloxypropylamine 5 (0.48 g, 1.0
mmol) in anhydrous chloroform (5 ml) and triethylamine (0.6 ml, 4
mmol) was added and the reaction was left to stir for an hour. TLC
(12% MeOH--CHCl.sub.3, developed in Molybdate) indicated that most
of the starting material had reacted to form product. The solution
was filtered through Celite with dichloromethane, acidified with
hydrochloric acid, and washed with distilled water (2.times.50 ml)
and brine (50 ml). The aqueous layers were back extracted with
dichloromethane (50 ml) and the combined organic layers were dried
over magnesium sulfate. The product was further purified my flash
column chromatography (0-7% MeOH--CHCl.sub.3) to yield 1.8 g
(69.0%) of N-(2,3-dimyristyloxypropyl) succinamide PEG.sub.2000
methyl ether 13.
[0317] It is to be understood that the above description is
intended to be illustrative and not restrictive. Many embodiments
will be apparent to those of skill in the art upon reading the
above description. The scope of the invention should, therefore, be
determined not with reference to the above description, but should
instead be determined with reference to the appended claims, along
with the full scope of equivalents to which such claims are
entitled. The disclosures of all Accession Nos., articles and
references, including patent applications, patents and PCT
publications, are incorporated herein by reference for all
purposes.
* * * * *
References