U.S. patent application number 14/970529 was filed with the patent office on 2016-07-21 for methods and compositions for enhancing delivery of double-stranded rna or a double-stranded hybrid nucleic acid to regulate gene expression in mammalian cells.
This patent application is currently assigned to Marina Biotech, Inc.. The applicant listed for this patent is Marina Biotech, Inc.. Invention is credited to Mohammad Ahmadian, Lishan Chen, Kunyuan Cui, Michael E. Houston, JR., Shu-Chih Chen Quay.
Application Number | 20160206749 14/970529 |
Document ID | / |
Family ID | 35907831 |
Filed Date | 2016-07-21 |
United States Patent
Application |
20160206749 |
Kind Code |
A1 |
Ahmadian; Mohammad ; et
al. |
July 21, 2016 |
METHODS AND COMPOSITIONS FOR ENHANCING DELIVERY OF DOUBLE-STRANDED
RNA OR A DOUBLE-STRANDED HYBRID NUCLEIC ACID TO REGULATE GENE
EXPRESSION IN MAMMALIAN CELLS
Abstract
Cholesterol moieties are linked to specific ends of
double-stranded RNA, preferably a small, interfering (si)RNA or to
a dsHybrid. The dsHybrid has one strand comprised of DNA and one
strand comprised of RNA. Preferably the sense strand is the DNA
strand and the antisense strand is the RNA strand of the dsHybrid.
The present invention is based upon the discovery that a
cholesterol moiety, if linked to a specific end or ends of the
sense or antisense strands of a siRNA, can enhance the delivery and
silencing efficiency of the siRNA directed against its target
message, in comparison with a corresponding, non-conjugated siRNA.
Conjugated siRNAs and dsHybrids of the invention are optionally
formulated with, or coordinately administered with, a secondary
delivery-enhancing agent, such as a delivery-enhancing peptide, to
enhance intracellular delivery and uptake of the conjugated siRNAs
or dsHybrid.
Inventors: |
Ahmadian; Mohammad;
(Bothell, WA) ; Cui; Kunyuan; (Bothell, WA)
; Chen; Lishan; (Bellevue, WA) ; Quay; Shu-Chih
Chen; (Seattle, WA) ; Houston, JR.; Michael E.;
(Sammamish, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Marina Biotech, Inc. |
New York |
NY |
US |
|
|
Assignee: |
Marina Biotech, Inc.
New York
NY
|
Family ID: |
35907831 |
Appl. No.: |
14/970529 |
Filed: |
December 15, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14566695 |
Dec 10, 2014 |
|
|
|
14970529 |
|
|
|
|
12870989 |
Aug 30, 2010 |
8940857 |
|
|
14566695 |
|
|
|
|
12013274 |
Jan 11, 2008 |
|
|
|
12870989 |
|
|
|
|
11107371 |
Apr 15, 2005 |
|
|
|
12013274 |
|
|
|
|
60564543 |
Apr 20, 2004 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C12Y 302/01023 20130101; C12N 2310/14 20130101; C12N 2310/3515
20130101; A61K 31/713 20130101; A61P 37/02 20180101; C12N 15/113
20130101; A61K 38/00 20130101; A61P 43/00 20180101; A61K 48/005
20130101; C12N 2320/30 20130101; A61P 35/00 20180101; A61K 47/554
20170801; A61P 31/12 20180101; C12N 15/1137 20130101; C12N 15/87
20130101; C07K 14/00 20130101; C12N 2320/32 20130101; C12N
2310/3513 20130101; C12N 15/111 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C12N 15/113 20060101 C12N015/113; A61K 48/00 20060101
A61K048/00; A61K 31/713 20060101 A61K031/713; A61K 45/06 20060101
A61K045/06 |
Claims
1.-96. (canceled)
97. A double-stranded (ds) RNA or a dsHybrid having a sense and an
anti-sense strand, each strand having a 5' and a 3' end, wherein:
(a) a cholesterol moiety is linked to the 5' end of the sense
strand and a cholesterol moiety is linked to the 5' end of the
antisense and wherein no cholesterol moiety is linked to the other
ends of the strands; (b) a cholesterol moiety is linked to the 3'
end of the antisense strand and wherein no cholesterol moiety is
linked to the other ends of the strands; (c) a cholesterol moiety
is linked to the 5' end of the sense strand and no cholesterol
moiety is linked to the other ends of the strands; (d) a
cholesterol moiety is linked to the 3' end of the sense strand and
no cholesterol moiety is linked to the other ends of the strands;
(e) a cholesterol moiety is linked to the 3' end of the sense
strand, a cholesterol moiety is linked to the 3' end of the
antisense strand and no cholesterol moiety is linked to the other
ends of the strands; a cholesterol moiety is linked to the 5' end
of the antisense strand and no cholesterol moiety is linked to the
other ends of the strands; (g) the 3' end of the sense strand is
connected to the 5' end of the antisense strand by means of a loop,
and wherein a cholesterol moiety is linked to the 3' end of the
antisense strand and wherein no cholesterol moiety is linked to the
other ends of the strands; and/or (h) the 3' end of the sense
strand is connected to the 5' end of the antisense strand by means
of a loop, wherein a cholesterol moiety is linked to the 5' end of
the sense strand and no cholesterol moiety is linked to the other
ends of the strands.
98. The dsRNA or dsHybrid of claim 97 wherein the dsRNA or dsHybrid
has a length of 19 to 21 base pairs.
99. The dsHybrid of claim 97 wherein the sense strand is a DNA
strand.
100. The dsRNA or dsHybrid of claim 97 wherein the cholesterol
moiety is selected from the group consisting of cholesterol,
sterol, any compound derived from cholesterol, chlolestanol,
ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid,
cortisol, corticosterone, .DELTA..sup.5-pregnenolone, progesterone,
deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone,
11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone
sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone,
tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone,
6.alpha.-methylpredisone,
9.alpha.-fluoro-16.alpha.-hydroxyprednisolone,
9.alpha.-fluoro-16.alpha.-methylprednisolone,
9.alpha.-fluorocortisol, testosterone, dihydrotestosterone,
androstenediol, androstenedione, androstenedione,
3.alpha.,5.alpha.-androstanediol, estrone, estradiol, estrogen,
spermidine cholesterol carbamate, N.sup.4-spermidine cholesteryl
carbamate, N.sup.4-spermidine cholesteryl carbamate di HCl salt,
N.sup.4-spermidine-7 dehydro cholesteryl carbamate, N4-spermine
cholesteryl carbamate, N,N bis(3-aminopropyl)cholesteryl carbamate,
N,N bis(6-aminohexyl)cholesteryl carbamate, N.sup.4-spermidine
dihydrocholesteryl carbamate, N.sup.4-spermidine lithocholic
carbamate methyl ester,
N.sup.1,N.sup.8-bis(3-aminopropyl-N.sup.4-spermidine cholesteryl
carbamate, N(N.sup.4-3aminopropylspermidine) cholesteryl carbamate,
N,N-bis(4-aminobutyl)cholesteryl carbamate, N.sup.4-spermidine
cholesteryl urea, N.sup.4-spermine cholesteryl urea,
N.sup.4-spermidine dihydro cholesteryl urea, N.sup.4-spermine
dihydro cholesteryl urea,
N,N-bis(N'-3-aminopropyl-N''4-aminobutyl)cholesteryl carbamate,
N4spermidine cholesteryl carboxamide, and
N-[N.sup.1,N.sup.4,N.sup.8-tris(3-aminopropyl)spermidine]cholesteryl
carbamate, lumisterol, cholic acid, desoxycholic acid,
chenodesoxycholic acid and lithocholic acid and derivative
thereof.
101. A method for producing double-stranded (ds) RNA comprising
hybridizing an RNA sense and an RNA antisense strand together, each
strand having a 5' and a 3'end, wherein: (a) a cholesterol moiety
is linked to the 5' end of the sense strand and a cholesterol
moiety is linked to the 5' end of the antisense and wherein no
cholesterol moiety is linked to the other ends of the strands; (b)
a cholesterol moiety is linked to the 3' end of the antisense
strand and wherein no cholesterol moiety is linked to the other
ends of the strands; (c) a cholesterol moiety is linked to the 5'
end of the sense strand and no cholesterol moiety is linked to the
other ends of the siRNA strands; (d) a cholesterol moiety is linked
to the 3' end of the sense strand and no cholesterol moiety is
linked to the other ends of the siRNA strands; (e) a cholesterol
moiety is linked to the 3' end of the sense strand, a cholesterol
moiety is linked to the 3' end of the antisense strand and no
cholesterol moiety linked to the other ends of the siRNA or
strands; (f) a cholesterol moiety is linked to the 5' end of the
antisense strand and no cholesterol moiety is linked to the other
ends of the dsRNA strands; (g) the 3' end of the sense strand is
connected to the 5' end of the antisense strand by means of a loop,
and wherein a cholesterol moiety is linked to the 3' end of the
antisense strand and wherein no cholesterol moiety is linked to the
other ends of the strands of the dsRNA a cholesterol moiety is
linked to the 5' end of the sense strand and no cholesterol moiety
is linked to the other ends of the dsRNA strands; (h) a cholesterol
moiety is linked to the 5' end of the sense strand and a
cholesterol moiety is linked to the 5' end of the antisense strand
and wherein no cholesterol moiety is linked to the other ends of
the strands; (i) a cholesterol moiety is linked to the 3' end of
the antisense strand and wherein no cholesterol moiety is linked to
the other ends of the strands; (j) a cholesterol moiety is linked
to the 5' end of the sense strand and no cholesterol moiety is
linked to the other ends of the strands; (k) a cholesterol moiety
is linked to the 3' end of the sense strand and no cholesterol
moiety is linked to the other ends of the strands; (l) a
cholesterol moiety is linked to the 3' end of the sense strand, a
cholesterol moiety is linked to the 3' end of the antisense strand
and no cholesterol moiety is linked to the other ends of the
strands; (m) a cholesterol moiety is linked to the 5' end of the
antisense strand and no cholesterol moiety is linked to the other
ends of the dsRNA strands; (n) the 3' end of the sense strand is
connected to the 5' end of the antisense strand by means of a loop,
and wherein a cholesterol moiety is linked to the 3' end of the
antisense strand and wherein no cholesterol moiety is linked to the
other ends of the strands of the dsHybrid; and/or (o) the 3' end of
the sense strand is connected to the 5' end of the antisense strand
by means of a loop, wherein a cholesterol moiety is linked to the
5' end of the sense strand and no cholesterol moiety linked to the
other ends of the dsHybrid strands.
102. The method of claim 101 wherein the dsRNA has a length of 19
to 21 base pairs.
103. The method of claim 101 wherein the cholesterol moiety is
selected from the group consisting of cholesterol, sterol, any
compound derived from cholesterol, chlolestanol, ergosterol,
stimastanol, stigmasterol, methyl-lithocholic acid, cortisol,
corticosterone, .DELTA..sup.5-pregnenolone, progesterone,
deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone,
11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone
sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone,
tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone,
6.alpha.-methylpredisone,
9.alpha.-fluoro-16.alpha.-hydroxyprednisolone,
9.alpha.-fluoro-16.alpha.-methylprednisolone,
9.alpha.-fluorocortisol, testosterone, dihydrotestosterone,
androstenediol, androstenedione, androstenedione,
3.alpha.,5.alpha.-androstanediol, estrone, estradiol, estrogen,
spermidine cholesterol carbamate, N.sup.4-spermidine cholesteryl
carbamate, N.sup.4-spermidine cholesteryl carbamate di HCl salt,
N.sup.4-spermidine-7 dehydro cholesteryl carbamate, N4-spermine
cholesteryl carbamate, N,N bis(3-aminopropyl)cholesteryl carbamate,
N,N bis(6-aminohexyl)cholesteryl carbamate, N.sup.4-spermidine
dihydrocholesteryl carbamate, N.sup.4-spermidine lithocholic
carbamate methyl ester,
N.sup.1,N.sup.8-bis(3-aminopropyl-N.sup.4-spermidine cholesteryl
carbamate, N(N.sup.4-3aminopropylspermidine)cholesteryl 6
carbamate, N,N-bis(4-aminobutyl)cholesteryl carbamate,
N.sup.4-spermidine cholesteryl urea, N.sup.4-spermine cholesteryl
urea, N.sup.4-spermidine dihydro cholesteryl urea, N.sup.4-spermine
dihydro cholesteryl urea,
N,N-bis(N'-3-aminopropyl-N''4-aminobutyl)cholesteryl carbamate,
N4spermidine cholesteryl carboxamide, and
N-[N.sup.1,N.sup.4,N.sup.8-tris(3-aminopropyl)spermidine]cholesteryl
carbamate, lumisterol, cholic acid, desoxycholic acid,
chenodesoxycholic acid and lithocholic acid and derivative
thereof.
104. A composition for enhancing delivery of a double-stranded (ds)
RNA or a dsHybrid into a cytoplasm of a mammalian target cell, said
composition comprising: (a) a double-stranded (ds) RNA or dsHybrid
and (b) one or more secondary delivery-enhancing agent(s) selected
from: (i) an aggregation inhibitory agent; (ii) a charge modifying
agent; (iii) a pH control agent; (iv) a degradative enzyme
inhibitory agent; (v) a mucolytic or mucus clearing agent; (vi) a
ciliostatic agent; (vii) a membrane penetration-enhancing agent
selected from a surfactant, a bile salt, a phospholipid additive, a
mixed micelle, a liposome, a carrier an alcohol, an enamine, an NO
donor compound, a long-chain amphipathic molecule, a small
hydrophobic penetration enhancer, sodium or a salicylic acid
derivative, a glycerol ester of acetoacetic acid, a cyclodextrin or
beta-cyclodextrin derivative, a medium-chain fatty acid, a
chelating agent, an amino acid or salt thereof, an N-acetylamino
acid or salt thereof, an enzyme degradative to a selected membrane
component, an inhibitor of fatty acid synthesis, and/or an
inhibitor of cholesterol synthesis; (viii) a delivery-enhancing
peptide; (ix) a vasodilator agent; (x) a selective
transport-enhancing agent; and (xi) a stabilizing delivery vehicle,
a carrier, a support, and/or a complex-forming species.
105. The composition of claim 104 wherein the dsRNA or dsHybrid has
a length of 19 to 21 base pairs.
106. The composition of claim 104 wherein the sense strand is a DNA
strand.
107. The composition of claim 104 wherein said double-stranded (ds)
RNA or dsHybrid comprises a sense and an anti-sense strand, each
strand having a 5' and a 3' end, wherein a cholesterol moiety is
linked to the 5' ends of the sense and antisense strands or to the
3' ends of the sense and antisense strands.
108. The composition of claim 107 wherein said cholesterol moiety
is selected from the group consisting of cholesterol, sterol, any
compound derived from cholesterol, chlolestanol, ergosterol,
stimastanol, stigmasterol, methyl-lithocholic acid, cortisol,
corticosterone, .DELTA..sup.5-pregnenolone, progesterone,
deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone,
11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone
sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone,
tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone,
6.alpha.-methylpredisone,
9.alpha.-fluoro-16.alpha.-hydroxyprednisolone,
9.alpha.-fluoro-16.alpha.-methylprednisolone,
9.alpha.-fluorocortisol, testosterone, dihydrotestosterone,
androstenediol, androstenedione, androstenedione,
3.alpha.,5.alpha.-androstanediol, estrone, estradiol, estrogen,
spermidine cholesterol carbamate, N.sup.4-spermidine cholesteryl
carbamate, N.sup.4-spermidine cholesteryl carbamate di HCl salt,
N.sup.4-spermidine-7 dehydro cholesteryl carbamate, N4-spermine
cholesteryl carbamate, N,N bis(3-aminopropyl)cholesteryl carbamate,
N,N bis(6-aminohexyl)cholesteryl carbamate, N.sup.4-spermidine
dihydrocholesteryl carbamate, N.sup.4-spermidine lithocholic
carbamate methyl ester,
N.sup.1,N.sup.8-bis(3-aminopropyl-N.sup.4-spermidine cholesteryl
carbamate, N(N.sup.4-3aminopropylspermidine)cholesteryl carbamate,
N,N-bis(4-aminobutyl)cholesteryl carbamate, N.sup.4-spermidine
cholesteryl urea, N.sup.4-spermine cholesteryl urea,
N.sup.4-spermidine dihydro cholesteryl urea, N.sup.4-spermine
dihydro cholesteryl urea,
N,N-bis(N'-3-aminopropyl-N''4-aminobutyl)cholesteryl carbamate,
N4spermidine cholesteryl carboxamide, and
N-[N.sup.1,N.sup.4,N.sup.8-tris(3-aminopropyl)spermidine]cholesteryl
carbamate, lumisterol, cholic acid, desoxycholic acid,
chenodesoxycholic acid and lithocholic acid and derivative
thereof.
109. The composition of claim 104 wherein the secondary
delivery-enhancing agent(s) include(s) a delivery-enhancing
peptide.
110. The composition of claim 104 wherein the delivery-enhancing
peptide comprises an amino acid sequence selected from:
TABLE-US-00006 (SEQ ID NO: 1) RKKRRQRRRPPQCAAVALLPAVLLALLAP; (SEQ
ID NO: 2) RQIKIWFQNRRMKWKK; (SEQ ID NO: 3)
GWTLNSAGYLLGKINLKALAALAKKIL; (SEQ ID NO: 4) KLALKLALKALKAALKLA;
(SEQ ID NO: 7) KLWSAWPSLWSSLWKP; (SEQ ID NO: 8)
AAVALLPAVLLALLAPRKKRRQRRRPPQ; (SEQ ID NO: 9)
LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR; (SEQ ID NO: 10)
RRRQRRKRGGDIMGEWGNEIFGAIAGFLG; (SEQ ID NO: 11)
KETWWETWWTEWSQPGRKKRRQRRRPPQ; (SEQ ID NO: 12)
GLGSLLKKAGKKLKQPKSKRKV; and (SEQ ID NO: 13)
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ
111. The composition of claim 104 wherein the delivery-enhancing
peptide is combined with or conjugated to the double-stranded (ds)
RNA or dsHybrid linked to the cholesterol moiety.
112. The composition of claim 104 which is effective to deliver the
double-stranded (ds) RNA or dsHybrid linked to the cholesterol
moiety to a target mammalian cell selected from pulmonary alveolar
cells, skin cells, hepatic cells, renal cells, pancreatic cells,
endothelial cells, nucleated blood cells, muscle cells, mammary
cells, peripheral or central nervous system (CNS) cells,
gastrointestinal cells, or tumor cells.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This U.S. non-provisional patent application was filed on
Dec. 15, 2015 and claims priority as a continuation to pending U.S.
patent application Ser. No. 14/566,695, filed Dec. 10, 2014, which
claims priority as a continuation to U.S. patent application Ser.
No. 12/870,989, filed Aug. 30, 2010, which claims priority as a
continuation to Ser. No. 12/013,274, filed Jan. 11, 2008, which
claims priority as a continuation to U.S. patent application Ser.
No. 11/107,371, filed Apr. 15, 2005, which claims the benefit of
U.S. Provisional Patent Application No. 60/564,543, filed Apr. 20,
2004. The entirety of U.S. patent application Ser. Nos. 14/566,695,
12/870,989, 12/013,274, and 11/107,371 and U.S. Provisional Patent
Application No. 60/564,543 are incorporated herein by
reference.
SEQUENCE LISTING
[0002] This application includes a Sequence Listing in electronic
format as a txt file entitled
"MRNA-01-2316USC4_2015-12-15_SEQLIST," which was created on Dec.
15, 2015 and which has a size of 8,315 bytes. The contents of txt
file "MRNA-01-2316USC4_2015-12-15_SEQLIST" are incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0003] RNA interference is the process of sequence-specific post
transcriptional gene silencing in cells initiated by
double-stranded RNA (dsRNA) that is homologous in sequence to a
portion of a targeted mRNA. Introduction of dsRNA into cells leads
to the destruction of the endogenous RNAs that share the same
sequence as the dsRNA. The dsRNA molecules are cleaved by an RNase
III family nuclease called Dicer into short-interfering RNAs
(siRNA), which are 19-23 nucleotides (nt) in length. The siRNAs are
incorporated into a multicomponent nuclease complex (RISC,
RNA-induced silencing complex), which identifies mRNA substrates
through their homology to the siRNA, binds to and destroys the
targeted mRNA. In mammalian cells, dsRNAs longer than 30 base pairs
can activate the dsRNA-dependent kinase PKR and
2'-5'-oligoadenylate synthetase, normally induced by interferon. By
virtue of its small size, synthetic siRNA avoids activation of the
interferon response. The activated PKR inhibits general translation
by phosphorylation of the translation factor eukaryotic initiation
factor 2a (eIF2.alpha.), while 2'-5'-oligoadenylate synthetase
causes nonspecific mRNA degradation via activation of RNase L.
[0004] In contrast to the nonspecific effect of long dsRNA, siRNA
can mediate selective gene silencing in the mammalian system
Hairpin RNA with a short loop and 19 to 27 base pairs in the stem
also selectively silences expression of genes that are homologous
to the sequence in the double-stranded stem. Mammalian cells can
convert short hairpin RNA into siRNA to mediate selective gene
silencing.
[0005] RISC mediates cleavage of single stranded RNA having
sequence complementary to the antisense strand of the siRNA duplex.
Cleavage of the target RNA takes place in the middle of the region
complementary to the antisense strand of the siRNA duplex.
[0006] Studies have shown that 21 nucleotide siRNA duplexes are
most active when containing two nucleotide 3'-overhangs.
Furthermore, complete substitution of one or both siRNA strands
with 2'-deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi
activity, whereas substitution of the 3'-terminal siRNA overhang
nucleotides with deoxy nucleotides (2'-H) was shown to be
tolerated.
[0007] Studies have shown that replacing the 3'-overhanging
segments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangs
with deoxyribonucleotides does not have an adverse effect on RNAi
activity. Replacing up to 4 nucleotides on each end of the siRNA
with deoxyribonucleotides has been reported to be well tolerated
whereas complete substitution with deoxyribonucleotides results in
no RNAi activity.
[0008] RNA interference is emerging as a promising means for
reducing the expression of specific gene products, and thus may be
useful for developing therapeutic drugs to treat viral infections,
cancers, autoimmune diseases, and other diseases and conditions
amenable to treatment by down-regulation of mRNA expression.
However, there remains an important need in the art for additional
tools and methods to design, produce, formulate, deliver, and use
siRNAs as therapeutic tools, including for therapies targeted to
specific tissues and cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 illustrates serum effects on cellular uptake of a
cholesterol-conjugated siRNA in complex with a delivery enhancing
agent (comprising a permeabilizing peptide, PN73), and on an
unconjugated siRNA in complex with PN73--expressed as percentage
uptake.
[0010] FIG. 2 illustrates serum effects on cellular uptake of a
cholesterol-conjugated siRNA in complex with PN73, and on an
unconjugated siRNA in complex with PN73--expressed as mean
fluorescence intensity (MFI).
[0011] FIG. 3 illustrates the effects of increasing concentrations
of serum on cellular uptake of a cholesterol-conjugated siRNA in
the presence or absence of a second delivery enhancing agent,
lipofectamine--expressed as percentage uptake.
[0012] FIG. 4 illustrates the effects of increasing concentrations
of serum on cellular uptake of a cholesterol-conjugated siRNA in
the presence or absence of a second delivery enhancing agent,
lipofectamine--expressed as MFI.
DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
[0013] The present invention fulfills these needs and satisfies
additional objects and advantages by providing double-stranded
nucleic acids conjugated to a cholesterol moiety to facilitate
delivery of the nucleic acids into a selected target cell or
tissue. In particular the present invention is directed towards
methods and compositions to administer double-stranded ribonucleic
acid to a mammal so as to effectuate transfection of the
double-stranded RNA into a desired tissue of the mammal. In certain
embodiments the double-stranded RNA has 30 or fewer nucleotides,
and is a short interfering RNA (siRNA).
[0014] It has been surprisingly discovered that selectively
conjugating a cholesterol moiety to a siRNA at selective ends of
the siRNA sense and/or antisense strands increases the silencing of
the targeted mRNA. For example, the following siRNA/cholesterol
moiety constructs increase the silencing effect of the targeted
mRNA in comparison to siRNA having no cholesterol conjugated to it:
[0015] 1. A siRNA construct having a cholesterol moiety linked to
the 5' end of the sense strand and the 5' end of the antisense, and
no cholesterol moiety at the other ends; [0016] 2. A siRNA
construct having a cholesterol moiety linked to the 3' end of the
antisense strand, and no cholesterol moiety linked to the other
ends of the siRNA strands; [0017] 3. A siRNA construct having
cholesterol moiety linked to the 5' end of the sense strand, and no
cholesterol moiety linked to the other ends of the siRNA strands;
[0018] 4. A siRNA construct having a cholesterol moiety linked to
the 3' end of the sense strand and no cholesterol moiety linked to
the other ends of the siRNA strands; [0019] 5. A siRNA construct
having a cholesterol moiety linked to the 3' end of the sense
strand, a cholesterol moiety linked to the 3' end of the antisense
strand and no cholesterol moiety linked to the other ends of the
siRNA strands; and [0020] 6. A siRNA construct having a cholesterol
moiety linked to the 5' end of the antisense strand and no
cholesterol moiety linked to the other ends of the siRNA
strands.
[0021] Thus, the constructs listed above are embodiments of the
present invention, as well as those constructs in which the ds
nucleic acid is a siHybrid in which the sense strand is a DNA
molecule.
[0022] The following constructs showed a progressively decreased
silencing of the targeted mRNA in comparison to a siRNA having no
cholesterol moieties conjugated to any of its ends: [0023] 1. A
siRNA construct having a cholesterol moiety linked to the 3' end of
the sense strand, a cholesterol moiety linked 5'end of the
antisense strand and no cholesterol moiety linked to the other ends
of the siRNA strands; [0024] 2. A siRNA construct having a
cholesterol moiety linked to the 3'end of the antisense strand, a
cholesterol moiety linked to the 5' end of the antisense strand and
no cholesterol moiety linked to the other ends of the siRNA
strands; [0025] 3. A siRNA construct having a cholesterol moiety
linked to the 5' end of the sense strand a cholesterol moiety
linked to the 3' end of the antisense strand and no cholesterol
moiety linked to the other ends of the siRNA strands; [0026] 4. A
siRNA construct having a cholesterol moiety linked to 5' end of the
sense strand, a cholesterol moiety linked to the 3' end of the
sense strand, and no cholesterol moiety linked to the other ends of
the siRNA strands; [0027] 5. A siRNA construct having a cholesterol
moiety linked to 5' end of the sense strand, a cholesterol moiety
linked to the 3' end of the antisense strand, a cholesterol moiety
linked to the 5' end of the antisense strand and no cholesterol
moiety linked to the 3' end of the sense strand; [0028] 6. A siRNA
construct having a cholesterol moiety linked to 5' end of the sense
strand, a cholesterol moiety linked to the 3' end of the sense
strand, a cholesterol moiety linked to the 3' end of the antisense
strand and no cholesterol moiety linked to the 5' end of the
antisense strand; [0029] 7. A siRNA construct having a cholesterol
moiety linked to 5' end of the sense strand, a cholesterol moiety
linked to the 3' end of the sense strand, a cholesterol moiety
linked to the 5' end of the antisense strand and no cholesterol
moiety linked to the 3' end of the antisense strand; [0030] 8. A
siRNA construct having a cholesterol moiety linked to 3' end of the
sense strand, a cholesterol moiety linked to the 3' end of the
sense strand, a cholesterol moiety linked to the 3' end of the
antisense strand, a cholesterol moiety linked to the 5' end of the
antisense strand, and no cholesterol moiety linked to the 5' end of
the sense strand; [0031] 9. A siRNA construct having a cholesterol
moiety on the 5' end of the sense strand, a cholesterol moiety on
the 3' end of the sense strand, a cholesterol moiety on the 3' end
of the antisense strand and a cholesterol moiety on the 5' end of
the antisense strand.
DEFINITIONS
[0032] As used herein, the term "inverted repeat" refers to a
nucleic acid sequence comprising a sense and an antisense element
positioned so that they are able to form a double stranded siRNA
when the repeat is transcribed. The inverted repeat may optionally
include a linker or a heterologous sequence such as a self-cleaving
ribozyme between the two elements of the repeat. The elements of
the inverted repeat have a length sufficient to form a double
stranded RNA. Typically, each element of the inverted repeat is
about 15 to about 100 nucleotides in length, preferably about 20-30
base nucleotides, preferably about 20-25 nucleotides in length,
e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in
length.
[0033] "Silencing" refers to partial or complete loss-of-function
through targeted inhibition of gene expression in a cell and may
also be referred to as "knock down". Depending on the circumstances
and the biological problem to be addressed, it may be preferable to
partially reduce gene expression. Alternatively, it might be
desirable to reduce gene expression as much as possible. The extent
of silencing may be determined by any method known in the art, some
of which are summarized in International Publication No. WO
99/32619. Depending on the assay, quantitation of gene expression
permits detection of various amounts of inhibition for example,
greater than 10%, 33%, 50%, 90%, 95% or 99%.
[0034] 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.
[0035] "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).
[0036] "Large double-stranded RNA" refers to any double-stranded
RNA having a size greater than about 40 base pairs (bp) for
example, larger than 100 bp or more particularly larger than 300
bp. The sequence of a large dsRNA may represent a segment of a mRNA
or the entire mRNA. The maximum size of the large dsRNA is not
limited herein. The double-stranded RNA may include modified bases
where the modification may be to the phosphate sugar backbone or to
the nucleoside. Such modifications may include a nitrogen or sulfur
heteroatom or any other modification known in the art.
[0037] The double-stranded structure may be formed by
self-complementary RNA strand such as occurs for a hairpin or a
micro RNA or by annealing of two distinct complementary RNA
strands.
[0038] "Overlapping" refers to when two RNA fragments have
sequences which overlap by a plurality of nucleotides on one
strand, for example, where the plurality of nucleotides (nt)
numbers as few as 2-5 nucleotides or by 5-10 nucleotides or
more.
[0039] "One or more dsRNAs" refers to dsRNAs that differ from each
other on the basis of sequence.
[0040] "Target gene or mRNA" refers to any gene or mRNA of
interest. Any of the genes previously identified by genetics or by
sequencing can be implemented as a target. Target genes or mRNA can
include developmental genes and regulatory genes, as well as
metabolic or structural genes or genes encoding enzymes. The target
gene may be expressed in cells in which a phenotype is being
investigated, or in an organism in a manner that directly or
indirectly impacts a phenotypic characteristic. The target gene may
be endogenous or exogenous. Such cells include any cell in the body
of an adult or embryonic animal or plant including gamete or any
isolated cell such as occurs in an immortal cell line or primary
cell culture.
[0041] In this specification and the appended claims, the singular
forms of "a", "an" and "the" include plural reference unless the
context clearly dictates otherwise.
[0042] "siRNA" means a small interfering RNA that is a short-length
double-stranded RNA that are not toxic in mammalian cells. The
length is not limited to 21 to 23 bp long. There is no particular
limitation in the length of siRNA as long as it does not show
toxicity. "siRNAs" can be, for example, 15 to 49 bp, preferably 15
to 35 bp, and more preferably 21 to 30 bp long. Alternatively, the
double-stranded RNA portion of a final transcription product of
siRNA to be expressed can be, for example, 15 to 49 bp, preferably
15 to 35 bp, and more preferably 21 to 30 bp long. The
double-stranded RNA portions of siRNAs in which two RNA strands
pair up are not limited to the completely paired ones, and may
contain nonpairing portions due to mismatch (the corresponding
nucleotides are not complementary), bulge (lacking in the
corresponding complementary nucleotide on one strand), and the
like. Nonpairing portions can be contained to the extent that they
do not interfere with siRNA formation. The "bulge" used herein
preferably comprise 1 to 2 nonpairing nucleotides, and the
double-stranded RNA region of siRNAs in which two RNA strands pair
up contains preferably 1 to 7, more preferably 1 to 5 bulges. In
addition, the "mismatch" used herein is contained in the
double-stranded RNA region of siRNAs in which two RNA strands pair
up, preferably 1 to 7, more preferably 1 to 5, in number. In a
preferable mismatch, one of the nucleotides is guanine, and the
other is uracil. Such a mismatch is due to a mutation from C to T,
G to A, or mixtures thereof in DNA coding for sense RNA, but not
particularly limited to them. Furthermore, in the present
invention, the double-stranded RNA region of siRNAs in which two
RNA strands pair up may contain both bulge and mismatched, which
sum up to, preferably 1 to 7, more preferably 1 to 5 in number.
[0043] The terminal structure of siRNA may be either blunt or
cohesive (overhanging) as long as siRNA enables to silence the
target gene expression due to its RNAi effect. The cohesive
(overhanging) end structure is not limited only to the 3' overhang
as reported by Tuschl et al. (ibid.), and the 5' overhanging
structure may be included as long as it is capable of inducing the
RNAi effect. In addition, the number of overhanging nucleotides is
not limited to the reported 2 or 3, but can be any numbers as long
as the overhang is capable of inducing the RNAi effect. For
example, the overhang may be 1 to 8, or 2 to 4 nucleotides. Herein,
the total length of siRNA having cohesive end structure is
expressed as the sum of the length of the paired double-stranded
portion and that of a pair comprising overhanging single-strands at
both ends. For example, in the case of 19 bp double-stranded RNA
portion with 4 nucleotide overhangs at both ends, the total length
is expressed as 23 bp. Furthermore, since this overhanging sequence
has low specificity to a target gene, it is not necessarily
complementary (antisense) or identical (sense) to the target gene
sequence. Furthermore, as long as the siRNA is able to maintain its
gene silencing effect on the target gene, it may comprise a low
molecular weight RNA (which may be a natural RNA molecule such as
tRNA, rRNA or viral RNA, or an artificial RNA molecule), for
example, in the overhanging portion at its one end.
[0044] In addition, the terminal structure of the "siRNA" is
necessarily the cut off structure at both ends as described above,
and may have a stem-loop structure in which ends of one side of
double-stranded RNA are connected by a linker RNA. The length of
the double-stranded RNA region (stem-loop portion) can be, for
example, 15 to 49 bp, preferably 15 to 35 bp, and more preferably
21 to 30 bp long. Alternatively, the length of the double-stranded
RNA region that is a final transcription product of siRNAs to be
expressed is, for example, 15 to 49 bp, preferably 15 to 35 bp, and
more preferably 21 to 30 bp long. Furthermore, there is no
particular limitation in the length of the linker as long as it has
a length so as not to hinder the pairing of the stem portion. For
example, for stable pairing of the stem portion and suppression of
the recombination between DNAs coding for the portion, the linker
portion may have a clover-leaf tRNA structure. Even though the
linker has a length that hinders pairing of the stem portion, it is
possible, for example, to construct the linker portion to include
introns so that the introns are excised during processing of
precursor RNA into mature RNA, thereby allowing pairing of the stem
portion. In the case of a stem-loop siRNA, either end (head or
tail) of RNA with no loop structure may have a low molecular weight
RNA. As described above, this low molecular weight RNA may be a
natural RNA molecule such as tRNA, rRNA or viral RNA, or an
artificial RNA molecule.
[0045] "Antisense RNA" is an RNA strand having a sequence
complementary to a target gene mRNA, and thought to induce RNAi by
binding to the target gene mRNA. "Sense RNA" has a sequence
complementary to the antisense RNA, and annealed to its
complementary antisense RNA to form siRNA. These antisense and
sense RNAs have been conventionally synthesized with an RNA
synthesizer.
[0046] As used herein, the term "RNAi construct" is a generic term
used throughout the specification to include small interfering RNAs
(siRNAs), hairpin RNAs, and other RNA species which can be cleaved
in vivo to form siRNAs. RNAi constructs herein also include
expression vectors (also referred to as RNAi expression vectors)
capable of giving rise to transcripts which form dsRNAs or hairpin
RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
Optionally, the siRNA include single strands or double strands of
siRNA.
[0047] An siHybrid molecule is a double-stranded nucleic acid that
has a similar function to siRNA. Instead of a double-stranded RNA
molecule, a siHybrid is comprised of an RNA strand and a DNA
strand. Preferably, the RNA strand is the antisense strand as that
is the strand that binds to the target mRNA. The siHybrid created
by the hybridization of the DNA and RNA strands have a hybridized
complementary portion and preferably at least one 3'overhanging
end.
[0048] A cholesterol moiety is a cholesterol molecule, sterol or
any compound derived from cholesterol including chlolestanol,
ergosterol, stimastanol, stigmasterol, methyl-lithocholic acid,
cortisol, corticosterone, .DELTA..sup.5-pregnenolone, progesterone,
deoxycorticosterone, 17-OH-pregnenolone, 17-OH-progesterone,
11-dioxycortisol, dehydroepiandrosterone, dehydroepiandrosterone
sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone,
tetrahydrocortisol, tetrahydrocortisone, cortisone, prednisone,
6.alpha.-methylpredisone,
9.alpha.-fluoro-16.alpha.-hydroxyprednisolone,
9.alpha.-fluoro-16.alpha.-methylprednisolone,
9.alpha.-fluorocortisol, testosterone, dihydrotestosterone,
androstenediol, androstenedione, androstenedione,
3.alpha.,5.alpha.-androstanediol, estrone, estradiol, estrogen,
spermidine cholesterol carbamate, N.sup.4-spermidine cholesteryl
carbamate, N.sup.4-spermidine cholesteryl carbamate di HCl salt,
N.sup.4-spermidine-7 dehydro cholesteryl carbamate, N4-spermine
cholesteryl carbamate, N,N bis(3-aminopropyl)cholesteryl carbamate,
N,N bis(6-aminohexyl)cholesteryl carbamate, N.sup.4-spermidine
dihydrocholesteryl carbamate, N.sup.4-spermidine lithocholic
carbamate methyl ester,
N.sup.1,N.sup.8-bis(3-aminopropyl-N.sup.4-spermidine cholesteryl
carbamate, N(N.sup.4-3aminopropylspermidine)cholesteryl carbamate,
N,N-bis(4-aminobutyl)cholesteryl carbamate, N.sup.4-spermidine
cholesteryl urea, N.sup.4-spermine cholesteryl urea,
N.sup.4-spermidine dihydro cholesteryl urea, N.sup.4-spermine
dihydro cholesteryl urea,
N,N-bis(N'-3-aminopropyl-N''4-aminobutyl)cholesteryl carbamate,
N4spermidine cholesteryl carboxamide, and
N-[N.sup.1,N.sup.4,N.sup.8-tris(3-aminopropyl)spermidine]cholesteryl
carbamate, lumisterol, cholic acid, desoxycholic acid,
chenodesoxycholic acid and lithocholic acid and derivatives thereof
(see, e.g., U.S. Pat. No. 6,331,524).
[0049] The following exemplary cholesterol-RNA constructs are
illustrative of various embodiments of the invention: [0050] 1. A
siRNA or siHybrid construct having a cholesterol moiety linked to
the 5' end of the sense strand and the 5'end of the antisense and
no cholesterol moiety at the other ends; [0051] 2. A siRNA or
siHybrid construct having a cholesterol moiety linked to the 3' end
of the antisense strand and no cholesterol moiety linked to the
other ends of the siRNA or siHybrid strands; [0052] 3. A siRNA or
siHybrid construct having cholesterol moiety linked to the 5' end
of the sense strand and no cholesterol moiety linked to the other
ends of the siRNA or siHybrid strands; [0053] 4. A siRNA or
siHybrid construct having a cholesterol moiety linked to the 3' end
of the sense strand and no cholesterol moiety linked to the other
ends of the siRNA or siHybrid strands; [0054] 5. A siRNA or
siHybrid construct having a cholesterol moiety linked to the 3' end
of the sense strand, a cholesterol moiety linked to the 3' end of
the antisense strand and no cholesterol moiety linked to the other
ends of the siRNA or siHybrid strands; and [0055] 6. A siRNA or
siHybrid construct having a cholesterol moiety linked to the 5' end
of the antisense strand and no cholesterol moiety linked to the
other ends of the siRNA or siHybrid strands.
[0056] In more detailed embodiments of the invention, a
cholesterol-conjugated siRNA or siHybrid is formulated with, or
delivered in a coordinate administration method with, one or more
secondary delivery-enhancing agent(s) that is/are further effective
to enhance delivery of the cholesterol-conjugated siRNA or siHybrid
into mammalian cells. Typically the second delivery-enhancing
agent(s) is/are effective to facilitate delivery of the
cholesterol-conjugated siRNA or siHybrid across the plasma membrane
and into the cytoplasm of a targeted mammalian cell. The targeted
cell may be any cell for which delivery of a cholesterol-conjugated
siRNA or siHybrid into the cell for regulation of gene expression
is desired. Exemplary target cells in this context include
pulmonary alveolar or other airway cells, skin cells, hepatic
cells, renal cells, pancreatic cells, endothelial cells, nucleated
blood cells (e.g., lymphocytes, monocytes, macrophages, or
dendritic cells), muscle cells (e.g., cardiac or smooth muscle
cells), mammary cells, peripheral or central nervous system (CNS)
cells, cells of the stomach or intestinal tract, tumor cells, and
other cells that are amenable to gene regulation for therapeutic
purposes according to the methods and compositions of the
invention.
[0057] In on exemplary embodiment, the cholesterol-conjugated siRNA
or siHybrid are targeted for delivery to mucosal epithelial cells,
for example nasal mucosal epithelial cells.
[0058] Within these and related aspects of the invention, the
secondary delivery-enhancing agent(s) may be selected from one or
any combination of the following:
[0059] (a) an aggregation inhibitory agent;
[0060] (b) a charge modifying agent;
[0061] (c) a pH control agent;
[0062] (d) a degradative enzyme inhibitory agent;
[0063] (e) a mucolytic or mucus clearing agent;
[0064] (f) a ciliostatic agent;
[0065] (g) a membrane penetration-enhancing agent selected from (i)
a surfactant, (ii) a bile salt, (iii) a phospholipid additive,
mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an
enamine, (vi) an NO donor compound, (vii) a long-chain amphipathic
molecule (viii) a small hydrophobic penetration enhancer; (ix)
sodium or a salicylic acid derivative; (x) a glycerol ester of
acetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin
derivative, (xii) a medium-chain fatty acid, (xiii) a chelating
agent, (xiv) an amino acid or salt thereof, (xv) an N-acetylamino
acid or salt thereof, (xvi) an enzyme degradative to a selected
membrane component, (xvii) an inhibitor of fatty acid synthesis, or
(xviii) an inhibitor of cholesterol synthesis; or (xix) any
combination of the membrane penetration enhancing agents recited in
(g)(i)-(xix);
[0066] (h) a delivery-enhancing peptide;
[0067] (i) a vasodilator agent;
[0068] (j) a selective transport-enhancing agent; and
[0069] (k) a stabilizing delivery vehicle, carrier, support or
complex-forming species with which the cholesterol-conjugated siRNA
or siHybrid is effectively combined, associated, contained,
encapsulated or bound resulting in stabilization of the siRNA or
siHybrid for enhanced delivery.
[0070] In additional aspects of the invention, the
delivery-enhancing agent(s) comprise(s) any one or any combination
of two or more of the foregoing delivery-enhancing agents recited
in (a)-(k), and the formulation of the cholesterol-conjugated siRNA
or siHybrid with the delivery-enhancing agents provides for
increased delivery of the cholesterol-conjugated siRNA or siHybrid
into the cytoplasm of target cells for gene regulation by the
cholesterol-conjugated siRNA or siHybrid.
[0071] Any one or combination of the foregoing secondary
delivery-enhancing agents may be added to a pharmaceutical
composition comprising a cholesterol-conjugated siRNA or siHybrid
as described herein, to yield a combinatorial formulation providing
greater delivery enhancement in comparison to intracellular
delivery of the cholesterol-conjugated siRNA or siHybrid without
the secondary delivery-enhancing agent(s).
[0072] Within coordinate administration methods of the invention,
the cholesterol-conjugated siRNA or siHybrid is administered to a
target cell, tissue, or individual in combination with one or more
secondary delivery-enhancing agents in a coordinate administration
protocol. Within these coordinate administration methods, the
cholesterol-conjugated siRNA or siHybrid is administered to the
same cell, tissue, or individual as the secondary
delivery-enhancing agent(s), prior to, simultaneous with, or after
administration of the secondary delivery-enhancing agent(s), which
similarly may be selected from any one or combination of the
following:
[0073] (a) an aggregation inhibitory agent;
[0074] (b) a charge modifying agent;
[0075] (c) a pH control agent;
[0076] (d) a degradative enzyme inhibitory agent;
[0077] (e) a mucolytic or mucus clearing agent;
[0078] (f) a ciliostatic agent;
[0079] (g) a membrane penetration-enhancing agent selected from (i)
a surfactant, (ii) a bile salt, (iii) a phospholipid additive,
mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an
enamine, (vi) an NO donor compound, (vii) a long-chain amphipathic
molecule (viii) a small hydrophobic penetration enhancer; (ix)
sodium or a salicylic acid derivative; (x) a glycerol ester of
acetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin
derivative, (xii) a medium-chain fatty acid, (xiii) a chelating
agent, (xiv) an amino acid or salt thereof, (xv) an N-acetylamino
acid or salt thereof, (xvi) an enzyme degradative to a selected
membrane component, (xvii) an inhibitor of fatty acid synthesis, or
(xviii) an inhibitor of cholesterol synthesis; or (xix) any
combination of the membrane penetration enhancing agents recited in
(g)(i)-(xix);
[0080] (h) a delivery-enhancing peptide;
[0081] (i) a vasodilator agent;
[0082] (j) a selective transport-enhancing agent; and
[0083] (k) a stabilizing delivery vehicle, carrier, support or
complex-forming species with which the cholesterol-conjugated siRNA
or siHybrid is effectively combined, associated, contained,
encapsulated or bound resulting in stabilization of the siRNA or
siHybrid for enhanced intracellular delivery. The coordinate
administration of the cholesterol-conjugated siRNA or siHybrid and
secondary delivery-enhancing agent(s) provides for increased uptake
of the cholesterol-conjugated siRNA or siHybrid into the cytoplasm
of targeted cells, typically enhancing gene regulation (e.g.,
increasing knockdown of mRNA translation to thereby reduce
expression of one or more selected protein(s), such as TNF-.alpha.,
in the target cell.
[0084] Additional detailed description pertaining to secondary
delivery-enhancing agents, for use within the instant invention is
provided, for example, in U.S. Provisional Patent Applications Nos.
60/612,121, filed Sep. 21, 2004; 60/667,835, filed Apr. 1, 2005;
60/612,285, filed Sep. 21, 2004; 60/667,871, filed Apr. 1, 2005;
60/613,416, filed Sep. 27, 2004; and 60/667,833, filed Apr. 1,
2005, each incorporated herein by reference.
[0085] Within exemplary embodiments of the invention, a
delivery-enhancing peptide is employed as the secondary
delivery-enhancing agent. The delivery-enhancing peptide may be
conjugated to, combinatorially formulated with, or coordinately
administered with, the cholesterol-conjugated siRNA or siHybrid to
enhance intracellular uptake of the cholesterol-conjugated siRNA or
siHybrid and improve gene regulation results achieved thereby.
Delivery-enhancing peptides in this context may include natural or
synthetic, therapeutically or prophylactically active, peptides
(comprised of two or more covalently linked amino acids), proteins,
peptide or protein fragments, peptide or protein analogs, peptide
or protein mimetics, and chemically modified derivatives or salts
of active peptides or proteins. Thus, as used herein, the term
"delivery-enhancing peptide" will often be intended to embrace all
of these active species, i.e., peptides and proteins, peptide and
protein fragments, peptide and protein analogs, peptide and protein
mimetics, and chemically modified derivatives and salts of active
peptides or proteins. Often, the delivery-enhancing peptide
comprises a mutein that is readily obtainable by partial
substitution, addition, or deletion of amino acids within a
naturally occurring or native (e.g., wild-type, naturally occurring
mutant, or allelic variant) peptide or protein sequence (e.g., a
sequence of a naturally occurring "cell penetrating peptide" or
peptide fragment of a native protein, such as a tight junction
protein). Additionally, biologically active fragments of native
peptides or proteins are included. Such mutant derivatives and
fragments substantially retain the desired cell penetrating or
other delivery-enhancing activity of the corresponding native
peptide or proteins. In the case of peptides or proteins having
carbohydrate chains, biologically active variants marked by
alterations in these carbohydrate species are also included within
the invention.
[0086] The delivery-enhancing peptides, proteins, analogs and
mimetics for use within the methods and compositions of the
invention are may be conjugated to, or formulated with, the
cholesterol-conjugated siRNA or siHybrid to yield a pharmaceutical
composition that includes a delivery-enhancing effective amount of
the delivery-enhancing peptide, protein, analog or mimetic (i.e.,
an amount of the peptide sufficient to detectably enhance
intracellular delivery of the cholesterol-conjugated siRNA or
siHybrid).
[0087] Exemplary delivery-enhancing peptides for use within the
methods and compositions of the invention include any one or
combination of the following peptides, or active fragments,
muteins, conjugates, or complexes thereof:
TABLE-US-00001 (SEQ ID NO: 1) RKKRRQRRRPPQCAAVALLPAVLLALLAP; (SEQ
ID NO: 2) RQIKIWFQNRRMKWKK; (SEQ ID NO: 3)
GWTLNSAGYLLGKINLKALAALAKKIL; (SEQ ID NO: 4) KLALKLALKALKAALKLA;
(SEQ ID NO: 7) KLWSAWPSLWSSLWKP; (SEQ ID NO: 8)
AAVALLPAVLLALLAPRKKRRQRRRPPQ; (SEQ ID NO: 9)
LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR; (SEQ ID NO: 10)
RRRQRRKRGGDIMGEWGNEIFGAIAGFLG; (SEQ ID NO: 11)
KETWWETWWTEWSQPGRKKRRQRRRPPQ; (SEQ ID NO: 12)
GLGSLLKKAGKKLKQPKSKRKV; and (SEQ ID NO: 13)
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ
[0088] Delivery-enhancing peptides of the invention may further
include various modifications known in the art, e.g., for modifying
the charge, membrane permeability, half-life, degradative
potential, reactivity (e.g., to form conjugates), immunogenicity,
or other desired properties of the subject peptide. Exemplary
modified delivery-enhancing peptides in this context may include,
for example, peptides modified by incorporation of one or more
selected amino- or carboxy-terminal chemical modifications. For
example, amino- and/or carboxy-terminal amide, BrAc, or maleimide
groups may be included, as exemplified by the modified
delivery-enhancing peptides shown in Table 1.
TABLE-US-00002 TABLE 1 Peptide Sequences Effects PN0028
RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide + (SEQ ID NO: 1) PN0058
RQIKIWFQNRRMKWKK-amide (SEQ ID NO: 2) + PN0064
BrAc-GWTLNSAGYLLGKINLKALAALAKKILamide + (SEQ ID NO: 3) PN0068
BrAc-KLALKLALKALKAALKLA-amide (SEQ ID NO: 4) + PN0069
GRKKRRQRRRPQ-amide (SEQ ID NO: 5) - PN0071 RRRRRRR (SEQ ID NO: 6) -
PN0228 NH2-KLWSAWPSLWSSLWKP-amide (SEQ ID NO: 7) +/- PN027
NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide + (SEQ ID NO: 8) PN202
NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRHRR-amide + (SEQ ID NO: 9) PN250
NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide + (SEQ ID NO: 10) PN183
NH2-KETWWETWWTEWSQPGRKKRRQRRRPPQ-amide + (SEQ ID NO: 11) PN283
Maleimide-GLGSLLKKAGKKLKQPKSKRKV-amide + (SEQ ID NO: 12) PN073
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide + (SEQ ID NO: 13)
Assay Medium Only
[0089] The + and - notations indicated in Table 1 for the listed
peptides relate to activity of the peptides to enhance permeation
of across epithelial monolayers--as determined by measurement of
peptide-mediated changes in trans-epithelial electrical resistance
(TEER). A + notation indicates that the subject peptide enhances
epithelial permeation of macromolecules. The peptides that exhibit
permeation-enhancing activity can be tested and selected according
to the methods herein to determine their utility for enhancing
delivery of cholesterol-conjugated siRNA or siHybrid into the
cytoplasm of targeted cells to enhance gene regulation.
[0090] The above disclosure generally describes the present
invention, which is further exemplified by the following examples.
These examples are described solely for purposes of illustration,
and are not intended to limit the scope of the invention. Although
specific terms and values have been employed herein, such terms and
values will likewise be understood as exemplary and non-limiting to
the scope of the invention.
EXAMPLE 1
Synthesis and Purification of Cholesterol-Labeled siRNA
[0091] Synthesis of Unmodified siRNAs:
[0092] Unmodified siRNAs were synthesized according to the general
strategy for solid-phase oligonucleotide synthesis. The syntheses
proceeded from the 3'- to 5'-direction [current protocols in
nucleic acid chemistry, chapter 3]. The first step involved
attachment of a mononucleoside/tide to the surface of an insoluble
solid support through a covalent bond. All unmodified siRNAs
described here were synthesized starting with a CPG-bound
deoxythymidine (purchased from Glen Research, Sterling, Va.). The
thymidine nucleoside is covalently attached to the solid support
through 3'-hydroxyl group using a base labile linker. Before chain
elongation can proceed, the terminal-protecting group
(dimethoxytrityl, DMT) on the nucleoside is removed. This exposes a
free 5'-OH group where the next nucleotide unit can be added. An
excess of reagents is used to force the coupling reaction to occur
on as many of the immobilized nucleotides as possible. After the
coupling reaction, excess reagents are washed away. The reaction is
followed by a c capping step, to block off non-extended sites, and
an oxidation step. The process of terminal-protecting group removal
and chain extension is then repeated using different bases until
the desired sequence has been assembled. Some or all of the
protecting groups may optionally be removed, and then the covalent
attachment to the support is hydrolyzed to release the product.
Removal of the protecting groups were carried out with 3:1 mixture
of concentrated ammonia:ethanol. After removal of any remaining
protecting groups, the oligonucleotide is ready for purification
and use.
[0093] RNA syntheses were carried out by Applied Biosystems 3400
using standard phosphoramidite chemistry. The corresponding
building blocks,
5'-dimethoxytrityl-N-benzoyladenosine-2'-O-(t-butyldimethylsilyl)-3'-[(2--
cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Bz-A-CE
phosphoramidite) (I),
5'-dimethoxytrityl-N-dimethylformamidine-guanosine,
2'-O-(t-butyldimethylsilyl)-3'-[(2-cyanoethyl)-(N,N-diisopropyl]-phosphor-
amidite (dmf-G-CE phosphoramidite) (II),
5'-dimethoxytrytiyl-N-acetylcytidine-2'-O-(t-butyldimethylsilyl)-3'-[(2-c-
yanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Ac-C-CE
phosphoramidite) (III),
5-dimethoxytrityluridine-2'-O-(t-butyldimethylsilyl)-3'-[(2-cyanoe-
thyl)-(N,N-diisopropyl)]-phosphoramidite (U-CE phosphoramidite)
(IV) and
5'-dimethoxytrityl-2'-deoxythymidine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)-
]-phosphoramidite (V) were purchased from Glen Research Inc.
(Sterling, Va.). For un-modified sequences, the syntheses started
on Controlled Pore glass (CPG) bound deoxytimidine (VI) (Applied
Biosystems, Foster City, Calif.) in 0.2 or 1.0 .mu.mol scale. Other
reagents and solvents were purchased from Glen Research (Sterling,
Va.) and/or Applied Biosystems (Foster City, Calif.).
##STR00001## ##STR00002##
Synthesis of 3'-Cholesteryl-Labeled siRNA
[0094] The synthesis of 3'-cholesteryl-labelled siRNAs was carried
out using the modified support strategy. In this method a new
modified solid phase synthesis support must be prepared for each
3'-reporter group or conjugate. The solid phase support for
attaching cholesteryl group to the 3'-termini of oligonucleotides
is commercially available. The synthesis of the
3'-cholesteryl-labelled oligonucleotides were accomplished using
1-dimethoyxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-
-glyceryl-2-O-succinoyl-long-chain-alkylamino-CPG (VII, Glen
Research, Sterling, Va.). The designed 21 nucleotide sequence was
then assembled on this modified solid support using standard
phosphoramidite protocols for RNA synthesis as described herein
above.
Synthesis of 5'-Cholesteryl-Labeled siRNA
[0095] A protected oligonucleotide with a free hydroxyl group at
the 5'-end, immobilized on the solid support, may easily be
obtained by solid phase synthesis using either methodologies
described herein above. The 5'-terminal hydroxyl can then be
reacted with phosphoramidites. Phosphoramidites often obtained from
a molecule having a hydroxyl functionality allow the direct
introduction of a functional group or ligand to the chain after
oxidation and deprotection. To incorporated a cholesteryl group to
the 5'-end of siRNA molecules,
dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-gl-
yceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite (VIII)
was purchased from Glen Research (Sterling, Va.). During the solid
support synthesis of siRNA, after the incorporation of the last
nucleoside/tide, the 5'-dimethoyxtrytyl protecting group was
cleaved and VIII was coupled to the grown chain
##STR00003##
1-dimethoyxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-
-glyceryl-2-O-succinoyl-long-chain-alkylamino-CPG (VII)
##STR00004##
dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethyleneglycol-gl-
yceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite (VIII)
Syntheses of 3' and 5'-Dicholesteryl-Labeled siRNAs
[0096] Syntheses of 3',5'-dicholesteryl-labeled siRNAs were
accomplished using a combination of the methods described above.
The synthesis of such a molecule started with using VII as the
"modified solid support", and elongation and incorporation of the
5'-cholesteryl moiety were carried out as described above.
EXAMPLE 2
Cholesterol-Enhanced Uptake of siRNA and Silencing of
Beta-Galactosidase mRNA Expression
[0097] Transfection of 9L/LacZ cells: [0098] Day 0: [0099] a) Take
saturated 9L/LacZ culture from T75 flask, detach cell and dilute
into 10 ml with complete medium (DMEM, 1.times.PS, 1.times.Na
Pyruvate, 1.times. NEAA). [0100] b) Further dilute the cell to
1:15, and seed 100 .mu.l into each 96 well, which should give 50%
confluence cell the next day for transfection. Remember to leave
the edge well empty and fill with 250 .mu.l water, do not stack up
plates in the incubator. [0101] c) Incubate overnight at 37.degree.
C., 5% CO.sub.2 incubator. [0102] Day 1: [0103] a) Prepare the
transfection complex in Opti-MEM, 50 .mu.l each well. [0104] b)
Dump the medium in plates, wash each well once with 200 .mu.l PBS
or Opti-MEM. [0105] c) Blot the plates dry completely with tissue
by inversion. [0106] d) Add the transfection mixture (50
.mu.l/well) into each well, add 250 .mu.l water into wells on the
edge to prevent wells from drying. [0107] e) Incubate for at least
3 hours at 37.degree. C., 5% CO.sub.2 incubator. [0108] f) Dump the
transfection mixture, replace with 100 .mu.l of complete medium
(DMEM, 1.times.PS, 1.times.Na Pyruvate, 1.times. NEAA).
.beta.-Gal/BCA Assay in 96 Well Format
Cell Lysis
[0108] [0109] a) Dump the medium, wash once with 200 .mu.l PBS,
blot the plate dry with inversion. [0110] b) Add 30 .mu.l lysis
buffer from .beta.-Gal Kit into each well. [0111] c) Freeze-Thaw
the cells twice to generate lysate.
.beta.-Gal Assay
[0111] [0112] a) Prepare assay mix (50 .mu.l 1.times. buffer, 17
.mu.l ONPG each well) [0113] b) Take new plate and add 65u1 assay
mix into each well. [0114] c) Add 10 .mu.l of cell lysate into each
well. There should be blank wells for subtraction of the background
activities. [0115] d) Incubate at 37.degree. C. for about 20
minutes, prevent long incubation which will use up all ONPG and
biased the high expression. [0116] e) Add 100 .mu.l of the Stop
solution. [0117] f) Measure the OD at 420 nm.
BCA Assay
[0117] [0118] a) Prepare BSA standard (150 ul per well), every
points should be duplicated on each plate. [0119] b) Put 145 .mu.l
of water into each well, add 5 ul of cell lysate into each well.
[0120] c) Prepare Assay Reagent (A:B:C: 25:24:1), mix right before
use. [0121] d) Add 150 .mu.l of Assay Reagent into each well.
[0122] e) Incubate at 37.degree. C. for about 20 minutes. [0123] f)
Measure the OD at 562 nm. Flow Cytometry Measurement of FITC/FAM
Conjugated siRNA [0124] a) After transfection, incubate cell for at
least 3 hours. [0125] b) Wash with 200 .mu.l PBS. [0126] c) Detach
cell with 15 .mu.l TE, incubate at 37.degree. C. [0127] d)
Re-suspend five wells with 30 .mu.l FACS solution (PBS with 0.5%
BSA, and 0.1% sodium Azide) [0128] e) Combine all five wells into a
tube. [0129] f) Add PI 5 .mu.l into each tube. [0130] g) Analyze
the cells with fluorescence activated cell sorting (FCAS) with BD
FACscan instrument according to manufacture's instruction.
Results
[0131] Cholesterol Conjugation of siRNA
[0132] The transfection was performed with either regular siRNA or
cholesterol-conjugated siRNA with lipofectamine (Invitrogen) on
9L/beta-gal cells. The siRNA was designed to specifically knock
down beta-galactosidase mRNA and activities are expressed as
percentage of beta-gal activities from control (transfected cells
by lipofectamine alone).
1. siRNA Sequence and Structure Information of
Cholesterol-Conjugated siRNA
TABLE-US-00003 (SEQ ID NO: 14)
C.U.A.C.A.C.A.A.A.U.C.A.G.C.G.A.U.U.U.dT.dT (Sense) (SEQ ID NO: 15)
A.A.A.U.C.G.C.U.G.A.U.U.U.G.U.G.U.A.G.dT.dT (Antisense)
Designation of Cholesterol Conjugated siRNA
[0133] A. regular sense or antisense strand
[0134] B. 5' end labeled sense strand
[0135] C. 3' end labeled sense strand
[0136] D. both ends labeled sense strand
[0137] E. 5' end labeled antisense strand
[0138] F. 3' end labeled antisense strand
[0139] G. both end labeled antisense strand
##STR00005## ##STR00006##
TABLE-US-00004 TABLE 1 Cholesterol siRNA Activities
Post-Transfection Duplexes Activity (% of control) Duplexes
Activity (% of control) AA 23.12 AG 29.87 BE 10.27 BF 32.02 AF
11.99 DA 33.99 BA 12.09 BG 46.39 CA 16.18 DF 65.4 CF 16.76 DE 77.12
AE 19.02 CG 77.80 CE 27.62 DG 98.84
[0140] Table 1 above provides results of transfection and mRNA
silencing experiments using the siRNA constructs made using sense
and antisense strands designated above. The transfection and
silencing assay results show cholesterol-enhanced delivery of
exemplary siRNAs of the invention, and demonstrate silencing of the
beta-galactosidase mRNA by the cholesterol-conjugated siRNAs. The
"Activity (% of control)" indicates the beta-galactosidase activity
remaining after the transfection. The lower the percentage, the
greater was the efficacy of the siRNA construct. The double letters
represent a double-stranded siRNA. Thus, the exemplary constructs,
BE, AF, BA, CA, CF, and AE are representative of the nature and
activity of cholesterol conjugated dsRNAs of the present invention.
These constructs show greater silencing efficacy than the
corresponding unconjugated siRNAs. siRNA constructs CE, AG, BF, DA,
BG, DF, DE, CG and DG showed lower efficacy than the unconjugated
siRNA construct AA.
[0141] AA is a siRNA construct with no cholesterol conjugated to
any of the ends of the sense or antisense RNA strands. This
construct was transfected into the cells resulting in silencing of
the beta-galactosidase mRNA so that 23.12% of the activity of the
beta-galactosidase mRNA remained.
[0142] BE is a siRNA construct having a cholesterol moiety linked
to the 5' end of the sense strand and a cholesterol moiety linked
to the 5'end of the antisense strand, and no cholesterol moiety
linked to the other ends of the siRNA. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that only 10.27% of the activity of the
beta-galactosidase mRNA remained. This is unexpectedly superior to
the unconjugated siRNA.
[0143] AF is a siRNA construct having a cholesterol moiety linked
to the 3' end of the antisense strand and no cholesterol moiety
linked to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that only 11.99% of the activity of the
beta-galactosidase mRNA remained. This is unexpectedly superior to
the unconjugated siRNA.
[0144] BA is a siRNA construct having a cholesterol moiety linked
to the 5' end of the sense strand and no cholesterol moiety linked
to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that only 12.09% of the activity of the
beta-galactosidase mRNA remained. This is unexpectedly superior to
the unconjugated siRNA.
[0145] CA is a siRNA construct having a cholesterol moiety linked
to the 3' end of the sense strand and no cholesterol moiety linked
to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that only 16.18% of the activity of the
beta-galactosidase mRNA remained. This is unexpectedly superior to
the unconjugated siRNA.
[0146] CF is a siRNA construct having a cholesterol moiety linked
to the 3' end of the sense strand, a cholesterol moiety linked to
the 3' end of the antisense strand, and no cholesterol moiety
linked to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that only 16.76% of the activity of the
beta-galactosidase mRNA remained. This is unexpectedly superior to
the unconjugated siRNA.
[0147] AE is a siRNA construct having a cholesterol moiety linked
to the 5' end of the antisense strand and no cholesterol moiety
linked to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that only 19.02% of the activity of the
beta-galactosidase mRNA remained. This is unexpectedly superior to
the unconjugated siRNA.
[0148] The constructs listed below showed lower ability to silence
the beta-galactosidase reporter than was determined for the
corresponding, unconjugated siRNA.
[0149] CE is a siRNA construct having a cholesterol moiety linked
to the 3' end of the sense strand, a cholesterol moiety linked
5'end of the antisense strand, and no cholesterol moiety linked to
the other ends of the siRNA strands. This construct was transfected
into the cells resulting in silencing the beta-galactosidase mRNA
so that 27.62% of the activity of the beta-galactosidase mRNA
remained. This silencing effect was lower than that observed for
the corresponding, unconjugated siRNA.
[0150] AG is a siRNA construct having a cholesterol moiety linked
to the 3'end of the antisense strand, a cholesterol moiety linked
to the 5' end of the antisense strand, and no cholesterol moiety
linked to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that 29.87% of the activity of the
beta-galactosidase mRNA remained. This silencing effect was lower
than that observed for the corresponding, unconjugated siRNA.
[0151] BF is a siRNA construct having a cholesterol moiety linked
to the 5' end of the sense strand, a cholesterol moiety linked to
the 3' end of the antisense strand, and no cholesterol moiety
linked to the other ends of the siRNA strands. This construct was
transfected into the cells resulting in silencing the
beta-galactosidase mRNA so that 32.02% of the activity of the
beta-galactosidase mRNA remained. This silencing effect was lower
than that observed for the corresponding, unconjugated siRNA.
[0152] DA is a siRNA construct having a cholesterol moiety linked
to 5' end of the sense strand, a cholesterol moiety linked to the
3' end of the sense strand, and no cholesterol moiety linked to the
other ends of the siRNA strands. This construct was transfected
into the cells resulting in silencing the beta-galactosidase mRNA
so that 33.99% of the activity of the beta-galactosidase mRNA
remained. This silencing effect was lower than that observed for
the corresponding, unconjugated siRNA.
[0153] BG is a siRNA construct having a cholesterol moiety linked
to 5' end of the sense strand, a cholesterol moiety linked to the
3' end of the antisense strand, a cholesterol moiety linked to the
5' end of the antisense strand, and no cholesterol moiety linked to
the 3' end of the sense strand. This construct was transfected into
the cells resulting in silencing the beta-galactosidase mRNA so
that 46.39% of the activity of the beta-galactosidase mRNA
remained. This silencing effect was lower than that observed for
the corresponding, unconjugated siRNA.
[0154] DF is a siRNA construct having a cholesterol moiety linked
to 5' end of the sense strand, a cholesterol moiety linked to the
3' end of the sense strand, a cholesterol moiety linked to the 3'
end of the antisense strand, and no cholesterol moiety linked to
the 5' end of the antisense strand. This construct was transfected
into the cells resulting in silencing the beta-galactosidase mRNA
so that 65.40% of the activity of the beta-galactosidase mRNA
remained. This silencing effect was lower than that observed for
the corresponding, unconjugated siRNA.
[0155] DE is a siRNA construct having a cholesterol moiety linked
to 5' end of the sense strand, a cholesterol moiety linked to the
3' end of the sense strand, a cholesterol moiety linked to the 5'
end of the antisense strand and no cholesterol moiety linked to the
3' end of the antisense strand. This construct was transfected into
the cells resulting in silencing the beta-galactosidase mRNA so
that 77.12% of the activity of the beta-galactosidase mRNA
remained. This silencing effect was lower than that observed for
the corresponding, unconjugated siRNA.
[0156] BG is a siRNA construct having a cholesterol moiety linked
to 3' end of the sense strand, a cholesterol moiety linked to the
3' end of the sense strand, a cholesterol moiety linked to the 3'
end of the antisense strand, a cholesterol moiety linked to the 5'
end of the antisense strand, and no cholesterol moiety linked to
the 5' end of the sense strand. This construct was transfected into
the cells resulting in silencing the beta-galactosidase mRNA so
that 77.80% of the activity of the beta-galactosidase mRNA
remained. This silencing effect was lower than that observed for
the corresponding, unconjugated siRNA.
[0157] DG is a siRNA construct having a cholesterol moiety on the
5' end of the sense strand, a cholesterol moiety on the 3' end of
the sense strand, a cholesterol moiety on the 3' end of the
antisense strand, and a cholesterol moiety on the 5' end of the
antisense strand. This construct was transfected into the cells
resulting in silencing the beta-galactosidase mRNA so that 98.84%
of the activity of the beta-galactosidase mRNA remained. This
silencing effect was lower than that observed for the
corresponding, unconjugated siRNA.
EXAMPLE 3
Serum Inhibition of Cholesterol-Enhanced siRNA Uptake, and Rescue
of Cholesterol Enhancement of Uptake by Additional
Delivery-Enhancing Agents
[0158] Human Monocyte Isolation and purity
[0159] Fresh human blood samples from healthy donors were purchased
from Golden West Biologicals (Temecula, Calif.). For isolation of
monocytes, blood samples were diluted with PBS at 1:1 ratio
immediately after receiving. Peripheral blood mononuclear cells
(PBMC) were first isolated by Ficoll (Amersham, Calif., USA)
gradient from whole blood. Then monocytes were further purified
from PBMCs using Miltenyi CD14 positive selection kit (MILTENYI
BIOTEC GmbH, Germany) by following the manufacturer's instructions.
The purity of the monocytes was greater than 95%, judged by flow
cytometry stained with anti-CD14 antibody (BD Biosciences, CA).
Purified human monocytes were maintained overnight in complete
media before induction and knockdown assay.
Flow Cytometry
[0160] Fluorescence activated cell sorting (FACS) analysis were
performed using Beckman Coulter FC500 cell analyzer (Fullerton,
Calif.). The instrument was adjusted according to the fluorescence
probes used (FAM or Cy5 for siRNA and FITC and PE for CD14).
Propidium iodide (Fluka, St. Lois, Mo.) and AnnexinV (R&D
systems, Minneapolis, Minn.) were used as indicators for cell
viability and cytotoxicity.
[0161] For siRNA uptake analysis, cells were washed with PBS,
treated with trypsin (attached cells only), and then analyzed by
flow cytometry. Uptake of the siRNA designated BA, described above,
was also measured by intensity of Cy5 or FAM fluorescence in the
cells and cellular viability assessed by addition of propidium
iodide or AnnexinV-PE. In order to differentiate the cellular
uptake from the membrane insertion of fluorescence labeled siRNA,
trypan blue was used to quench the fluorescence on the cell
membrane surface.
TABLE-US-00005 TABLE 2 Higher MFI with PN73 compared with
cholesterol siRNA alone Cholesterol Unconjugated siRNA Serum siRNA
alone with 20 .mu.M PN73 0 24.8 32.9 5% 1.55 11.5 10% 1.34 6.39 20%
1.19 5.85
[0162] The data in Table 2 show that the presence of serum
significantly reduces cellular uptake of the siRNA conjugated to a
cholesterol moiety according to the invention. Serum also inhibits
unconjugated siRNA uptake in the presence of an exemplary
delivery-enhancing peptide, PN73
(KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-amide; SEQ ID NO: 13), but to
a lesser extent than the inhibition noted for the
cholesterol-conjugated siRNA.
[0163] FIGS. 1 and 2 illustrate the effects of 5% serum on cellular
uptake of a cholesterol-conjugated siRNA according to the invention
in complex with a permeabilizing peptide delivery enhancing agent,
PN73 (cholesterol siRNA+PN73), and on an unconjugated siRNA in
complex with PN73 (siRNA+PN73). For these and related uptake
assays, cholesterol-conjugated siRNA and siRNA/PN73 complex were
transfected into human monocytes in Opti-MEM.RTM. media
(Invitrogen) as described above, with serum added in fixed or
varied concentration(s). The final concentration of siRNA for both
cholesterol and complex were 0.2 .mu.M. The uptake efficiency and
Mean fluorescence intensity were assessed by flow cytometry. The
cellular uptake values shown in FIGS. 1 and 2 were determined with
variation of PN73 concentrations in the presence of a fixed, 5%
concentration of serum.
[0164] FIGS. 3 and 4 illustrate the effects of varying
concentrations of serum on cellular uptake of a
cholesterol-conjugated siRNA in the presence or absence of a second
delivery enhancing agent, lipofectamine, as determined by flow
cytometry.
[0165] The foregoing studies demonstrate that
cholesterol-conjugation of siRNAs can significantly enhance their
cellular uptake. However, uptake of cholesterol-conjugated siRNAs
can be substantially diminished or even eliminated by the presence
of serum. This is likely due to binding of the cholesterol moiety
with serum proteins--inhibiting the ability of the
cholesterol-bound siRNAs to enter target cells. In the presence of
a selected delivery enhancing agent, Lipofectamine, this inhibitory
effect of serum on cholesterol-siRNA uptake can be effectively
diminished. In addition, the presence of a different kind of
delivery enhancing agent, exemplified by the permeabilizing peptide
PN73, can also mediate rescue of siRNA delivery blocked by serum.
More specifically, the addition of a permeabilizing peptide to a
delivery formulation comprising a siRNA conjugated to a cholesterol
moiety reduces the inhibitory effects of serum on cholesterol-siRNA
uptake in a dose dependent manner. This discovery indicates that,
although cholesterol conjugation to siRNA alone may not optimize
siRNA delivery, additional delivery-enhancing agents including, but
not limited to, Lipofectamine and PN73, can further enhance siRNA
delivery to mammalian cells and tissues in vitro and in vivo.
[0166] Although the foregoing invention has been described in
detail by way of example for purposes of clarity of understanding,
it will be apparent to the artisan that certain changes and
modifications may be practiced within the scope of the appended
claims which are presented by way of illustration not limitation.
In this context, various publications and other references have
been cited within the foregoing disclosure for economy of
description. Each of these references is incorporated herein by
reference in its entirety for all purposes. It is noted, however,
that the various publications discussed herein are incorporated
solely for their disclosure prior to the filing date of the present
application, and the inventors reserve the right to antedate such
disclosure by virtue of prior invention.
Sequence CWU 1
1
21129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic HIV virus and Homo sapiens construct 1Arg Lys Lys Arg Arg
Gln Arg Arg Arg Pro Pro Gln Cys Ala Ala Val 1 5 10 15 Ala Leu Leu
Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 20 25 216PRTDrosophila
melanogaster 2Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys
Trp Lys Lys 1 5 10 15 327PRTUnknown OrganismDescription of Unknown
Organism Human, swine, or mouse, and wasp venom 3Gly Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala
Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Lys
Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys 1 5 10
15 Leu Ala 512PRTHuman immunodeficiency virus 5Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg Pro Gln 1 5 10 67PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Arg
Arg Arg Arg Arg Arg Arg 1 5 716PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Lys Leu Trp Ser Ala Trp Pro
Ser Leu Trp Ser Ser Leu Trp Lys Pro 1 5 10 15 828PRTArtificial
SequenceDescription of Artificial Sequence Synthetic HIV virus and
Homo sapiens construct 8Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu
Ala Leu Leu Ala Pro 1 5 10 15 Arg Lys Lys Arg Arg Gln Arg Arg Arg
Pro Pro Gln 20 25 933PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Leu Leu Glu Thr Leu Leu Lys
Pro Phe Gln Cys Arg Ile Cys Met Arg 1 5 10 15 Asn Phe Ser Thr Arg
Gln Ala Arg Arg Asn His Arg Arg Arg His Arg 20 25 30 Arg
1029PRTInfluenza virus 10Arg Arg Arg Gln Arg Arg Lys Arg Gly Gly
Asp Ile Met Gly Glu Trp 1 5 10 15 Gly Asn Glu Ile Phe Gly Ala Ile
Ala Gly Phe Leu Gly 20 25 1128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 11Lys Glu Thr Trp Trp Glu Thr
Trp Trp Thr Glu Trp Ser Gln Pro Gly 1 5 10 15 Arg Lys Lys Arg Arg
Gln Arg Arg Arg Pro Pro Gln 20 25 1222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Gly
Leu Gly Ser Leu Leu Lys Lys Ala Gly Lys Lys Leu Lys Gln Pro 1 5 10
15 Lys Ser Lys Arg Lys Val 20 1336PRTHomo sapiens 13Lys Gly Ser Lys
Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys 1 5 10 15 Lys Arg
Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys 20 25 30
Val Leu Lys Gln 35 1421DNAArtificial SequenceDescription of
Combined DNA/RNA Molecule Synthetic siRNA oligonucleotide construct
14cuacacaaau cagcgauuut t 211521DNAArtificial SequenceDescription
of Combined DNA/RNA Molecule Synthetic siRNA oligonucleotide
construct 15aaaucgcuga uuuguguagt t 211621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic siRNA
oligonucleotide construct 16cuacacaaau cagcgauuut t
211721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic siRNA oligonucleotide construct 17cuacacaaau
cagcgauuut t 211821DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic siRNA oligonucleotide construct
18cuacacaaau cagcgauuut t 211921DNAArtificial SequenceDescription
of Combined DNA/RNA Molecule Synthetic siRNA oligonucleotide
construct 19aaaucgcuga uuuguguagt t 212021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic siRNA
oligonucleotide construct 20aaaucgcuga uuuguguagt t
212121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic siRNA oligonucleotide construct 21aaaucgcuga
uuuguguagt t 21
* * * * *