U.S. patent application number 12/750622 was filed with the patent office on 2010-09-23 for lipopeptides for delivery of nucleic acids.
This patent application is currently assigned to MDRNA, INC.. Invention is credited to Roger C. Adami, Michael E. Houston, Rachel E. Johns.
Application Number | 20100240731 12/750622 |
Document ID | / |
Family ID | 40404245 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240731 |
Kind Code |
A1 |
Adami; Roger C. ; et
al. |
September 23, 2010 |
LIPOPEPTIDES FOR DELIVERY OF NUCLEIC ACIDS
Abstract
Lipopeptide compounds comprising a peptide having 2 to 100 amino
acid residues, and having a lipophilic group attached to at least
one terminus of the peptide or to at least one amino acid residue
of the peptide, and salts and uses thereof. The lipophilic group
may be attached to the N-terminus, C-terminus or both termini of
the peptide. The lipophilic group may be attached to at least one
interal amino acid residue (i.e., an amino acid residue that is not
the N-terminus or the C-terminus amino acid residue of the
peptide). The lipophilic group may be attached to either termini or
both and at least one internal amino acid residue.
Inventors: |
Adami; Roger C.; (Bothell,
WA) ; Houston; Michael E.; (Sammamish, WA) ;
Johns; Rachel E.; (Shoreline, WA) |
Correspondence
Address: |
Eckman Basu LLP
2225 E. Bayshore Road, Suite 200
Palo Alto
CA
94303-3220
US
|
Assignee: |
MDRNA, INC.
Bothell
WA
|
Family ID: |
40404245 |
Appl. No.: |
12/750622 |
Filed: |
March 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2008/078627 |
Oct 2, 2008 |
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12750622 |
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60976894 |
Oct 2, 2007 |
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Current U.S.
Class: |
514/44A ;
530/324; 530/326; 530/327; 530/328; 530/329; 530/330; 530/331 |
Current CPC
Class: |
A61K 47/6455 20170801;
C07K 14/001 20130101; C07K 7/08 20130101; A61K 47/544 20170801;
A61P 29/00 20180101; A61P 3/00 20180101; A61K 47/42 20130101; A61P
35/00 20180101; C12N 15/113 20130101; C12N 2310/14 20130101; A61K
47/543 20170801; A61K 31/713 20130101; C12N 15/87 20130101; A61K
48/0033 20130101; A61K 48/00 20130101; A61P 31/12 20180101; A61K
48/0041 20130101; C07K 14/00 20130101 |
Class at
Publication: |
514/44.A ;
530/324; 530/326; 530/327; 530/328; 530/329; 530/330; 530/331 |
International
Class: |
A61K 31/713 20060101
A61K031/713; C07K 14/00 20060101 C07K014/00; C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06; A61P 35/00 20060101
A61P035/00; A61P 3/00 20060101 A61P003/00; A61P 29/00 20060101
A61P029/00; A61P 31/12 20060101 A61P031/12; C07K 5/10 20060101
C07K005/10; C07K 5/08 20060101 C07K005/08 |
Claims
1. The compound of claim 1, comprising the structure shown in
Formula I:
R.sup.3-J-{NR.sup.N--CR.sup.1R.sup.2--(C.dbd.O)}.sub.n--Z--R.sup.4
Formula I wherein R.sup.1 is independently, for each occurrence, a
non-hydrogen side chain of an amino acid;
{NR.sup.N--CR.sup.1R.sup.2--(C.dbd.O)}.sub.n is a central peptide;
R.sup.2 and R.sup.N are independently of one another hydrogen, or
an organic group consisting of carbon, oxygen, nitrogen, sulfur,
and hydrogen atoms, and having from 1 to 20 carbon atoms, or
C(1-22)alkyl, (6-12)cycloalkyl, (6-12)cycloalkylalkyl,
C(3-18)alkenyl, C(3-18)alkynyl, C(1-5)alkanoyl, C(1-5)alkanoyloxy,
C(1-5)alkoxy, C(1-5)alkoxy-C(1-5)alkyl, C(1-5) alkoxy-C(1-5)alkoxy,
C(1-5)alkyl-amino-C(1-5)alkyl-, C(1-5)dialkyl-amino-C(1-5)alkyl-,
nitro-C(1-5)alkyl, cyano-C(1-5)alkyl, aryl-C(1-5)alkyl,
4-biphenyl-C(1-5)alkyl, carboxyl, or hydroxyl; R.sup.3 and R.sup.4
are independently of one another, a lipophilic tail derived from a
naturally-occurring or synthetic lipid, phospholipid, glycolipid,
triacylglycerol, glycerophospholipid, sphingolipid, ceramide,
sphingomyelin, cerebroside, or ganglioside, wherein the tail may
contain a steroid, or an organic group consisting of carbon,
oxygen, nitrogen, sulfur, and hydrogen atoms, and having from 1 to
20 carbon atoms, or a substituted or unsubstituted C(1-22)alkyl,
C(6-12)cycloalkyl, C(6-12)cycloalkyl-alkyl, C(3-18)alkenyl,
C(3-18)alkynyl, C(1-5)alkoxy-C(1-5)alkyl, or a sphinganine,
(2R,3R)-2-amino-1,3-octadecanediol, icosasphinganine, sphingosine,
phytosphingosine, cis-4-sphingenine, or a ceramide; Z is NH, O, or
a linker comprising a maleimido, thioether, amide, cysteamide,
cysteine, thiol, or a disulfide group, or a polyethyleneoxide or
polypropyleneoxide group comprising 1-400 atoms, or a linker
comprising 1-200 atoms selected from the group of C, H, F, Cl, Br,
N, O, S, Si, and P; J is (C.dbd.O), O, or a linker comprising a
maleimido, thioether, amide, cysteamide, cysteine, thiol, or
disulfide group, or a polyethyleneoxide or polypropyleneoxide group
comprising 1-400 atoms, or a linker comprising 1-200 atoms selected
from the group of C, H, F, Cl, Br, N, O, S, Si, and P; and wherein
n is 2 to 100.
2. The compound of claim 1, wherein the amino acid sequence of the
central peptide is selected from the group consisting of SEQ ID
NOs: 1-300 and 350-356.
3. The compound of claim 1, wherein the amino acid sequence of the
central peptide is selected from the group consisting of
GALFLAFLAAALX.sub.aaLMGLWX.sub.aaQX.sub.aaKKKRKV (SEQ ID NO: 291),
WSQPKKKRKV (SEQ ID NO: 292), WWHHKKRRCCRRKKHHWW (SEQ ID NO:94),
X.sub.aaWSQPKKKRKVX.sub.aa (SEQ ID NO:293),
WX.sub.aaQPKKKRKX.sub.aa (SEQ ID NO:294)
X.sub.aaX.sub.aaQPKKKRKV(SEQ ID NO:295),
LIRLWX.sub.aaHLIHIWFQX.sub.aaRRLKWKKK (SEQ ID NO: 297), QNRRLKWKKK
(SEQ ID NO:298), X.sub.aaQNRRLKWKKKX.sub.aa (SEQ ID NO:299), and
QX.sub.aaRRLKWKKK (SEQ ID NO:300), where X.sub.aa is independently,
for each occurance, L, A, Q, H, E, R, or K.
4. The compound of claim 1, wherein a side chain is linked to the
central peptide, wherein the side chain contains a releasing
functional group selected from 3,5-diiodo-tyrosine,
1-methylhistidine, 2-methylbutanoic acid, 2-o-anisylpropanoic acid,
meso-tartaric acid, 4,6-dimethylpyrimidinamine, p-phthalic acid,
creatinine, butanoic acid, N,N-dimethyl-1-naphthylamine, pentanoic
acid, 4-methylpentanoic acid, N-methylaniline, 1,10-phenanthroline,
3-pyridinecarboxylic acid, hexanoic acid, propanoic acid,
4-animobenzoic acid, 2-methylpropanoic acid, heptanoic acid,
octanoic acid, cyclohexanecarboxylic acid, quinoline,
3-quinolinamine, 2-aminobenzoic acid, 4-pyridinecarboxylic acid,
nonanic acid, melamine, 8-quinolinol, trimethylacetic acid,
6-methoxyquinoline, 4-(methylamino)benzoic acid, p-methylaniline,
3-(methylamino)benzoic acid, malic acid, N-ethylaniline,
2-benzylpyridine, 3,6-dinitrophenol, N,N-dimethylaniline,
2,5-dimethylpiperazine, p-phenetidine, 5-methylquinoline,
2-phenylbenzimidazole, pyridine, picolinic acid,
3,5-diiodityrosine, p-anisidine, 2-(methylamino)benzoic acid,
2-thiazolamine, glutaric acid, adipic acid, isoquinoline, itaconic
acid, o-phthalic acid, benzimidazole, piperazine, heptanedioic
acid, acridine, phenanthridine, succinic acid, methylsuccinic acid,
4-methylquinoline, 3-methylpyridine, 7-isoquinolinol, malonic acid,
methymalonic acid, 2-methylquinoline, 2-ethylpyridine,
2-methylpyridine, 4-methylpyridine, histamine, histidine, maleic
acid, cis-1,2-cyclohexanediamine, 3,5-dimethylpyridine,
2-ethylbenzimidazole, 2-methylbenzimidazole, cacodylic acid,
perimidine, citric acid, isocitric acid, 2,5-dimethylpyridine,
papaverine, 6-hydroxy-4-methylpteridine, L-thyroxine,
3,4-dimethylpyridine, methoxypyridine,
trans-1,2-cyclohexanediamine, 2,5-pyridinediamine,
l-1-methylhistidine, l-3-methylhistidine, 2,3-dimethylpyridine,
xanthopterin, 1,2-propanediamine, N,N-diethylaniline, alloxanic
acid, 2,6-dimethylpyridine, L-carnosine, 2-pyridinamine,
N-b-alanylhistidine, pilocarpine, 1-methylimidazol, 1H-imidazole,
2,4-dimethylpyridine, 4-nitrophenol, 2-nitrophenol, tyrosineamide,
5-hydroxxyquinazoline, 1,1-cyclopropanedicarboxylic acid,
2,4,6-trimethylpyridine, veronal, 2,3-dichlorophenol,
1,2-ethanediamine, 1-isoquinolinamine, and combinations
thereof.
5. A pharmaceutical composition comprising a nucleic acid agent and
one or more compounds according to claim 1, or
pharmaceutically-acceptable salts thereof.
6. The composition of claim 5, wherein the nucleic acid agent is a
usiRNA.
7. The composition of claim 5, wherein the nucleic acid agent is an
mdRNA.
8. The composition of claim 5, wherein the nucleic acid agent is an
siRNA.
9. The composition of claim 5, wherein the one or more compounds is
(C20)-(H).sub.8(R).sub.8(H).sub.8K-NH-(C20) (SEQ ID NO: 13).
10. The composition of claim 5, further comprising a non-cationic
lipid.
11. The composition of claim 5, further comprising a polymeric
lipid.
12. A method for delivering an interfering RNA agent to a cell
comprising preparing a composition according to claim 5 and
treating a cell with the composition.
13. A method for inhibiting expression of a gene in a cell
comprising preparing a composition according to claim 5 and
treating a cell with the composition.
14. A method for inhibiting expression of a gene in a mammal
comprising preparing a composition according to claim 5 and
administering the composition to the mammal.
15. A method for treating the signs and symptoms of a disease,
disorder, or condition in a subject selected from cancer, a
proliferative disease, disorder, or condition, a metabolic disease,
disorder, or condition, an inflammatory disease, disorder, or
condition, and a viral infection by providing a composition of
claim 5 and administering the composition to the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of prior PCT
International Application No. PCT/US2008/078627, filed Oct. 2,
2008, which claims the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Application No. 60/976,894, filed Oct. 2, 2007, each of
which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
herewith via EFS-Web as an ASCII file created on Mar. 25, 2010,
named MDR-07-23USB1_SeqList.txt, which is 134,532 bytes in size,
and is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to novel drug delivery enhancing
agents including lipids that are useful for delivering various
molecules to cells. This invention provides a range of compounds,
compositions, formulations, methods and uses of such agents
directed ultimately toward drug delivery, therapeutics, and the
diagnosis and treatment of diseases and conditions, including those
that respond to modulation of gene expression or activity in a
subject. More specifically, this invention relates to compounds,
liposomes, lamellar vesicles, emulsions, micelles, suspensions,
particles, solutions and other forms of delivery enhancing
compositions and formulations, as well as therapeutic methods and
uses for these delivery materials.
BACKGROUND
[0004] The delivery of a therapeutic compound to a subject can be
impeded by limited ability of the compound to reach a target cell
or tissue, or by restricted entry or trafficking of the compound
within cells. Delivery of a therapeutic material is in general
restricted by membranes of cells. These barriers and restrictions
to delivery can result in the need to use much higher
concentrations of a compound than is desirable to achieve a result,
which brings the risk of toxic effects and side effects.
[0005] One strategy for delivery is to improve transport of a
compound into cells using lipid or polymeric carrier molecules.
These materials can take advantage of mechanisms that exist for
selective entry into a cell, while still excluding exogenous
molecules such as nucleic acids and proteins. For example, a
cationic lipid may interact with a drug agent and provide contact
with a cell membrane. Lipid molecules can also be organized into
liposomes or particles as carriers for drug agents. Liposomal drug
carriers can protect a drug molecule from degradation while
improving its uptake by cells. Also, liposomal drug carriers can
encapsulate or bind certain compounds by electrostatic and other
interactions, and may interact with negatively charged cell
membranes to initiate transport across a membrane.
[0006] The understanding of regulatory RNA and the development of
RNA interference (RNAi), RNAi therapy, RNA based drugs, antisense
therapy, and gene therapy, among others, has increased the need for
effective means of introducing active nucleic acid agents into
cells. In general, nucleic acids are stable for only limited times
in cells or plasma. However, nucleic acid-based agents can be
stabilized in compositions and formulations which may then be
dispersed for cellular delivery.
[0007] What is needed are compositions and formulations for
intracellular and in vivo delivery of a nucleic acid agent for use,
ultimately, as a therapeutic, which maintain cytoprotection and
relatively low toxicity. Furthermore, there is a need for
compositions and methods to deliver double-stranded RNA to cells to
produce the response of RNA interference. Moreover, there is a need
for compositions and methods for delivery of interfering RNAs to
selected cells, tissues, or compartments to modulate gene
expression in a manner that will alter a phenotype or disease
state.
BRIEF SUMMARY
[0008] This invention satisfies these needs and fulfills additional
objects and advantages by providing a range of novel compounds,
compositions, formulations and methods that employ an interfering
nucleic acid or precursor thereof in combination with various
components including lipopeptides, lipids, and natural or synthetic
polymers.
[0009] In some embodiments, this invention includes a peptide
having 2 to 100 amino acid residues and a lipophilic group attached
to at least one terminus of the peptide, or to at least one amino
acid residue, and salts and uses thereof. The lipophilic group may
be attached to the N-terminus, C-terminus or both termini of the
peptide. The lipophilic group may be attached to at least one
interal amino acid residue (i.e., an amino acid residue that is not
the N-terminus or the C-terminus amino acid residue of the
peptide). The lipophilic group may be attached to either termini or
both and at least one internal amino acid residue.
[0010] This summary, taken along with the detailed description of
the invention, as well as the appended examples and claims as a
whole encompasses the disclosure.
DETAILED DESCRIPTION
[0011] This invention relates generally to the fields of RNA
interference, delivery of RNA therapeutics, and to the chemistry of
lipids. More particularly, this invention relates to compositions
and formulations for ribonucleic acids, and their uses for
medicaments and for delivery as therapeutics. This invention
relates generally to methods of using ribonucleic acids in RNA
interference for gene-specific inhibition of gene expression in
mammals.
[0012] This invention provides a range of compositions,
formulations and methods which include an interfering nucleic acid
or a precursor thereof in combination with various components
including lipopeptides, lipids, lipoid moieties, and natural or
synthetic polymers.
[0013] In some aspects, this invention provides novel compositions
to facilitate the delivery of nucleic acids including RNAi-inducing
agents, antisense RNA agents, or DNA to cells, tissues, organs, and
in living animals, for example, mammals and humans.
Lipopeptides
[0014] This invention provides a range of lipopeptides for delivery
and administration of interfering-RNA agents or antisense RNAs. The
lipopeptides can be cationic, or non-cationic. As used herein,
non-cationic includes neutral and anionic. Cationic lipopeptides
may have one or more cationic sites.
[0015] Lipopeptides of this disclosure may be formed by
substituting an nucleic acid delivery-enhancing or lipoid group at
the N-terminus or the C-terminus of a peptide, or both termini of
the peptide. In some aspects, the nucleic acid delivery or lipoid
group may be linked to the alpha-carbon of one or more an amino
acid residues of the peptide or to one or more amine groups, each
amine group being adjacent to the alpha-carbon of an amino acid
residue of the peptide, or a combination of the N-terminus,
C-terminus, alpha-carbon of one or more an amino acid residues of
the peptide or to one or more amine groups, each amine group being
adjacent to the alpha-carbon of an amino acid residue of the
peptide. In some embodiments, the peptide core may include two or
more sequential amino acids, or a peptide of 2 to 100 amino acid
residues (or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100
amino acid residues).
[0016] In some aspects, this invention provides a range of
lipopeptide structures as shown in Formula I:
R.sup.3-J-{NR.sup.N--CR.sup.1R.sup.2--(C.dbd.O)}.sub.n--Z--R.sup.4
Formula I
[0017] wherein [0018] R.sup.1 is independently, for each
occurrence, a non-hydrogen side chain of an amino acid; [0019]
{NR.sup.N--CR.sup.1R.sup.2--(C.dbd.O)}.sub.n is a central peptide;
[0020] R.sup.2 and R.sup.N are independently of one another
hydrogen, or an organic group consisting of carbon, oxygen,
nitrogen, sulfur, and hydrogen atoms, and having from 1 to 20
carbon atoms, or C(1-22)alkyl, (6-12)cycloalkyl,
(6-12)cycloalkylalkyl, C(3-18)alkenyl, C(3-18)alkynyl, [0021]
C(1-5)alkanoyl, C(1-5)alkanoyloxy, C(1-5)alkoxy,
C(1-5)alkoxy-C(1-5)alkyl, C(1-5)alkoxy-C(1-5)alkoxy,
C(1-5)alkyl-amino-C(1-5)alkyl-, C(1-5)dialkyl-amino-C(1-5)alkyl-,
nitro-C(1-5)alkyl, cyano-C(1-5)alkyl, aryl-C(1-5)alkyl,
4-biphenyl-C(1-5)alkyl, carboxyl, or hydroxyl; [0022] R.sup.3 and
R.sup.4 are independently of one another, a lipophilic tail derived
from a naturally-occurring or synthetic lipid, phospholipid,
glycolipid, triacylglycerol, glycerophospholipid, sphingolipid,
ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the
tail may contain a steroid, or an organic group consisting of
carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, and having
from 1 to 20 carbon atoms, or a substituted or unsubstituted
C(1-22)alkyl, C(6-12)cycloalkyl, C(6-12)cycloalkyl-alkyl,
C(3-18)alkenyl, C(3-18)alkynyl, C(1-5)alkoxy-C(1-5)alkyl, or a
sphinganine, (2R,3R)-2-amino-1,3-octadecanediol, icosasphinganine,
sphingosine, phytosphingosine, cis-4-sphingenine, or a ceramide;
[0023] Z is NH, O, or a linker comprising a maleimido, thioether,
amide, cysteamide, cysteine, thiol, or a disulfide group, or a
polyethyleneoxide or polypropyleneoxide group comprising 1-400
atoms, or a linker comprising 1-200 atoms selected from the group
of C, H, F, Cl, Br, N, O, S, Si, and P; [0024] J is (C.dbd.O), O,
or a linker comprising a maleimido, thioether, amide, cysteamide,
cysteine, thiol, or disulfide group, or a polyethyleneoxide or
polypropyleneoxide group comprising 1-400 atoms, or a linker
comprising 1-200 atoms selected from the group of C, H, F, Cl, Br,
N, O, S, Si, and P; and
[0025] n is from 2 to 100.
[0026] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide is capable of binding nucleic
acids.
[0027] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide may be an amphipathic amino acid
sequence. For example, the peptide may have a plurality of
non-polar or hydrophobic amino acid residues that form a
hydrophobic sequence domain or motif which may be linked to a
plurality of charged amino acid residues that form a charged
sequence domain or motif, yielding an amphipathic peptide.
[0028] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide may be Poly-Lys-Trp, 4:1, M.sub.W
20,000-50,000; Poly-Orn-Trp, 4:1, M.sub.W 20,000-50,000; fragments
or variants of mellitin protein, and fragments or variants of a
histone protein, e.g., histone H1, histone H2A, histone H2B,
histone H3 or histone H4.
[0029] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide has multiple cationic sites or
multiple positively-charged residues.
[0030] The central peptides may comprise from about 2 to about 45
positively-charged residues; from about 3 to about 45
positively-charged residues; from about 4 to about 45
positively-charged residues; from about 5 to about 45
positively-charged residues; from about 10 to about 45
positively-charged residues; from about 15 to about 45
positively-charged residues; from about 20 to about 45
positively-charged residues; from about 25 to about 45
positively-charged residues; from about 30 to about 45
positively-charged residues; from about 35 to about 45
positively-charged residues; from about 40 to about 45
positively-charged residues.
[0031] The peptides may be thiolylated substantially polylysine
peptides. They may comprise at least 3, 4, 5, 6, 7, 8 or so thiol
groups or may have only two thiol groups.
[0032] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide is arginine (Arg).sub.n (SEQ ID NO:
341), homoarginine (homoArg).sub.n (side chain
--(CH.sub.2).sub.4NH(C.dbd.NH)NH.sub.2), norarginine (norArg).sub.n
(side chain --(CH.sub.2).sub.2NH(C.dbd.NH)NH.sub.2),
nor-norarginine (nornorArg).sub.n (side chain
--(CH.sub.2)NH(C.dbd.NH)NH.sub.2), ornithine (Orn).sub.n, lysine
(Lys).sub.n, (SEQ ID NO: 342), histidine (His).sub.n (SEQ ID NO:
343), where n is 2 to 100.
[0033] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide is (Arg,His).sub.n, (Lys,His).sub.n,
(Arg,Lys).sub.n, His-(Arg).sub.n-His (SEQ ID NO: 344),
His-(Lys).sub.n-His (SEQ ID NO: 345),
(His).sub.n-(Arg).sub.n-(His).sub.n (SEQ ID NO: 346), or
(His).sub.n-(Lys).sub.n-(His).sub.n(SEQ ID NO: 347), where n is 2
to 100.
[0034] Examples of the central peptide of lipopeptides of this
disclosure include those shown in Table 1.
TABLE-US-00001 TABLE 1 Peptide Structures for Lipopeptides AMINO
ACID SEQUENCE SEQ ID NO: (R).sub.n where n = 1-20 1 (K).sub.n where
n = 1-20 2 (R).sub.n(K).sub.m where n and m are independtly 3 for
each occurence 1-20 (RH).sub.n or (HR).sub.n where n = 1-20 4 &
348 [(RH).sub.n(HR).sub.m] where n and m are 5 independtly for each
occurence 1-20 (KH).sub.n or (HK).sub.n where n = 1-20 6 & 349
[(KH).sub.n(HK).sub.m] where n and m are 7 independtly for each
occurence 1-20 H(K).sub.nH where n = 1-20 8 H(R).sub.nH where n =
1-20 9 (H).sub.8(R).sub.8 10 (H).sub.4(R).sub.8 11
(H).sub.4(R).sub.4(K).sub.4 12 (H).sub.8(R).sub.8(H).sub.8(K) 13
(H).sub.4(R).sub.5(H).sub.3(K).sub.3 14 HHHHHKHHHKKKHKHKKK 15
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 16
KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ 17 AQKKEGKKRKRSRKESYSVYVYKVLKQ
18 KRSRKESYSVYVYKVLKQ 19 ESYSVYVYKVLKQ 20 YKVLKQ 21 RVIRWFQNKRSKDKK
22 GALFLGFLGAAGSTMGAWSQPKSKRKV 23 RQIKIWFQNRRMKWKK 24
RQIKIWFQNRRMKWKK (ALL D-AMINO ACIDS) 25 GWTLNSAGYLLKINLKALAALAKKIL
26 LLNQLAGRMIPKWSQKSKRKV 27 TLDHVLDHVQTWSQKSKRKV 28
SYFILRRRRKRFPYFFTDVRVAA 29 RRRRRRRRRR 30 RRRRRRRR (all D-amino
acids) 31 KETWWETWWTEWSQPGRKKRRQRRRPPQ 32 GRPRESGKKRKRKRLKP 33
KSYSVYVYKVLKQ 34 ESYSVYVYRVLRQ 35 RSYSVYVYRVLRQ 36 QKLVKYVYVSYSE 37
ESYSVYVYKVLKQ (all D-amino acids) 38 ASYSVYVYAVLAQ 39 QKLVKYVYVSYSE
(all D-amino acids) 40 RRRRRRESYSVYVYKVLKQ 41 ESYSVYVYKVLKQRRRRRR
42 RRRRRRRQIKIWFQNRRMKWKK 43 RQIKIWFQNRRMKWKKRRRRRR 44
KTKIESLKEHGRRRRRR 45 MDVNPTLLFLKVPAQNAISTTFPYTRRRRRR 46
GLFEALLELLESLWELLLEARRRRRR 47 LLNQLAGRMIPKRRRRRR 48
TLDHVLDHVQTRRRRRR 49 GLFGAIAGFIENGWEGMIDGRRRRRR 50
KETWWETWWTERRRRRR 51 HHHHHHHHHHRRRRRR 52 AAVALLPAVLLALLAPRRRRRR 53
KVLKQ 54 KRRQRRR 55 DAATATRGRSAASRPTERPRAPARSASRPRRPVD 56
VTVLALGALAGVGVG 57 GALFLGWLGAAGSTMGA 58 MGLGLHLLVLAAALQGA 59
LGTYTQDFNKFHTFPQTAIGVGAP 60 GWTLNSAGYLLKINLKALAALAKKIL 61
TPPKKKRKVEDPKKKK 62 KLALKLALKALKAALKLA 63 GLFGAIAGFIENGWEG 64
FFGAVIGTIALGVATA 65 FLGFLLGVGSAIASGV 66 GVFVLGFLGFLATAGS 67
GAAIGLAWIPYFGPAA 68 ACTCPYCKDSEGRGSGDPGKKKQHICHIQGCGKVYGK 69
TSHLRAHLRWHTGERPFMC ACTCPNCKDGEKRSGEQGKKKHVCHIPDCGKTFRKTS 70
LLRAHVRLHTGERPFVC ACTCPNCKEGGGRGTNLGKKKQHICHIPGCGKVYGKTS 71
HLRAHLRWHSGERPFVC ACSCPNCREGEGRGSNEPGKKKQHICHIEGCGKVYGKT 72
SHLRAHLRWHTGERPFIC RCTCPNCTNEMSGLPPIVGPDERGRKQHICHIPGCERLY 73
GKASHLKTHLRWHTGERPFLC TCDCPNCQEAERLGPAGVHLRKKNIHSCHIPGCGKVYG 74
KTSHLKAHLRWHTGERPFVC RCTCPNCKAIKHGDRGSQHTHLCSVPGCGKTYKKTSHL 75
RAHLRKHTGDRPFVC PQISLKKKIFFFIFSNFRGDGKSRIHICHLCNKTYGKTS 76
HLRAHLRGHAGNKPFAC WWETWKPFQCRICMRNFSTRQARRNHRRRHR 77
GKINLKALAALAKKIL 78 RVIRVWFQNKRCKDKK 79
GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ 80 GEQIAQLIAGYIDIILKKKKSK
81 KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 82
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 83 RKESYSVYVYKVLKQ 84
KKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ 85 VTKAQKKDGKKRKRSRKESYSVYVYKVLKQ
86 AQKKDGKKRKRSRKESYSVYVYKVLKQ 87 KDGKKRKRSRKESYSVYVYKVLKQ 88
KKRKRSRKESYSVYVYKVLKQ 89 SYSVYVYKVLKQ 90 VYVYKVLKQ 91
KGSKKAVTKAQKKEGKKRKRSRKESYSVYVYKVLKQ 92 WWHHKKRRC 93
WWHHKKRRCCRRKKHHWW 94 GRKKRRQRRRPPQ 95 KKKRKV 96 KKKRKVKKKRKV 97
GRKKRRC 98 RRRPPQC 99 CGRKKRR 100 CRRRPPQ 101 WKKKKC 102 CWKKKK 103
CRRRPPQH 104 CRRRPPQ 105 CKKRRQH 106 CRR 107 CRRR 108 CRRRR 109
CRRRRR 110 CKK 111 CKKK 112 CKKKK 113 CKKKKK 114 CRRRRWW 115 CRRRWW
116 CRRWW 117
CKKWW 118 CKKKWW 119 CKKKKWW 120 CWHHRRKK 121 CRRKKHHWW 122 CKKRRW
123 CKKRRHW 124 CKKRRHHW 125 CKKRRQ 126 KKRRQC 127 CGRKKRR 128
GRKKRRC 129 CGRKKRRQ 130 QGRKKRRC 131 CRRH 132 CRRRH 133 CRRRRH 134
CRRRRRH 135 CKKH 136 CKKKH 137 CKKKKH 138 CKKKKKH 139 HWKKRRC 140
CHWKKRR 141 PPHRRRC 142 CPPHRRR 143 GRKKRRVURRRPPQ 144
GRKKRRVURRKKRG 145 RRRPPQVUPPRRR 146 RRKKRGVUGRKKRR 147
QPPRRRVURRRPPQ 148 WKKKKVUKKKKW 149 KKKKWVUWKKKK 150
HQPPRRRVURRRPPQH 151 QPPRRRVURRRPPQ 152 HQRRKKVUKKRRQH 153 RRVURR
154 RRRVURRR 155 RRRRVURRRR 156 RRRRRVURRRRR 157 KKVUKK 158
KKKVUKKK 159 KKKKVUKKKK 160 KKKKKVUKKKKK 161 WWRRRRVURRRRWW 162
WWRRRVURRRWW 163 WWRRVURRWW 164 WWKKVUKKWW 165 WWKKKVUKKKWW 166
WWKKKKVUKKKKWW 167 KKRRHHWVUWHHRRKK 168 WWHHKKRRVURRKKHHWW 169
WRRKKVUKKRRW 170 WHRRKKVUKKRRHW 171 WHHRRKKVUKKRRHHW 172
QRRKKVUKKRRQ 173 KKRRQVUQRRKK 174 RRKKRGVUGRKKRR 175 GRKKRRVURRKKRG
176 QRRKKRGVUGRKKRRQ 177 QGRKKRRVURRKKRGQ 178 HRRVURRH 179
HRRRVURRRH 180 HRRRRVURRRRH 181 HRRRRRVURRRRRH 182 HKKVUKKH 183
HKKKVUKKKH 184 HKKKKVUKKKKH 185 HKKKKKVUKKKKKH 186 HWKKRRVURRKKWH
187 RRKKWHVUHWKKRR 188 PPHRRRVURRRHPP 189 RRRHPPVUPPHRRR 190
WWHHKKRRGGRRKKHHWW 191 WWHHKKRR 192 YYHHKKRR 193 RRKKHHYY 194
YYHHKKRRCCRRKKHHYY 195 YYHHKKRRVRRKKHHYY 196 WWRR 197 RRWW 198
CWRRRWC 199 CWWRRRWWC 200 CWRRRRWC 201 CWRRHHRRWC 202 CWWRRRRWWC
203 CWWRRHHRRWWC 204 CWRRRRRWC 205 VQAAIDYING 206 CWWRRRRRWWC 207
WWRRCCRRWW 208 WWRRVRRWW 209 YYRR 210 RRYY 211 CYRRRYC 212
CYYRRRYYC 213 CYRRRRYC 214 CYRRHHRRYC 215 CYYRRRRYYC 216
CYYRRHHRRYYC 217 CYRRRRRYC 218 CYYRRRRRYYC 219 CYYRRRRYYC 220
YYRRCCRRYY 221 YYRRVRRYY 222 WWRRHH 223 HHRRWW 224 WWRRHHCCHHRRWW
225 WWRRHHVHHRRWW 226 YYRRHH 227 HHRRYY 228 YYRRHHCCRRHHYY 229
YYRRHHVRRHHYY 230 WWRRR 231 RRRWW 232 WWRRRCCRRRWW 233 WWRRRVRRRWW
234 YYRRR 235 RRRYY 236 YYRRRCCRRRYY 237 YYRRRVRRRYY 238 WWRRRHH
239 HHRRRWW 240 WWRRRHHCCHHRRRWW 241 WWRRRHHVHHRRRWW 242 YYRRRHH
243
HHRRRYY 244 YYRRRHHCCRRRHHYY 245 YYRRRHHVRRRHHYY 246 WWRRRR 247
RRRRWW 248 WWRRRRCCRRRRWW 249 WWRRRRVRRRRWW 250 YYRRRR 251 RRRRYY
252 YYRRRRCCRRRRYY 253 YYRRRRVRRRRYY 254 WWRRRRHH 255 HHRRRRWW 256
WWRRRRHHCCHHRRRRWW 257 WWRRRRHHVHHRRRRWW 258 YYRRRRHH 259 HHRRRRYY
260 YYRRRRHHCCRRRRHHYY 261 YYRRRRHHVRRRRHHYY 262
WWHHKKRRWVWRRKKHHWW 263 WWHHRRC 264 WWHHRRVRRHHWW 265 WWHHHRRRC 266
CWWHHHRRRC 267 CWWWHHHHRRR 268 CWWWKKRRR 269 CKKKWRRW 270 CWRRRWRR
271 CWWHHKKRRC 272 WWCHHKKCRR 273 WWHHCKKRRC 274 CWWHHKKCRR 275
WWHHCCKKRR 276 RRWWKKHHC 277 CWWHHKKKKC 278 CWWHHRRRRC 279 CRRRRHHC
280 CHHKKKKC 281 CHHRRRRC 282 CYYRRRRHHC 283 CYYKKKKHHC 284
CWK.sub.nC where n is from 1-20 285 CHWK.sub.nC where n is from
1-20 286 AAABX.sub.aaYX.sub.aaQWLX.sub.aaX.sub.aaX.sub.aaGPX.sub.aa
where X.sub.aa 287 is any amino acid CK.sub.nC where n is from 1-20
288 CHK.sub.nHC where n is from 1-20 289
[0035] Examples of the central peptide of lipopeptides of this
disclosure include those shown in Table 2.
TABLE-US-00002 TABLE 2 Peptide Structures for Lipopeptides AMINO
ACID SEQUENCE SEQ ID NO: GALFLAFLAAALSLMGLWSQPKKKRKV 290
GALFLAFLAAALX.sub.aaLMGLWX.sub.aaQX.sub.aaKKKRKV 291 WSQPKKKRKV 292
X.sub.aaWSQPKKKRKVX.sub.aa 293 WX.sub.aaQPKKKRKX.sub.aa 294
X.sub.aaX.sub.aaQPKKKRKV 295 LIRLWSHLIHIWFQNRRLKWKKK 296
LIRLWX.sub.aaHLIHIWFQX.sub.aaRRLKWKKK 297 QNRRLKWKKK 298
X.sub.aaQNRRLKWKKKX.sub.aa 299 QX.sub.aaRRLKWKKK 300 where X.sub.aa
in Table 2 is independently, for each occurrence, L, A, Q, H, E, R,
or K.
[0036] The amino acid residues of this disclosure may be D- or
L-stereocenters.
[0037] Non-cationic lipopeptides can be prepared where, for
example, the central peptide contains only leucine, valine,
alanine, serine, or combinations thereof.
[0038] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide may be cell or tissue specific
targeting peptide. For example, the peptide may be a ligand or
fragment thereof that interacts with a cell surfact receptor or a
peptide the selectively binds to lipids, peptides, sugars and
combinations thereof on the cell surface of a particular cell or
tissue type. The central peptide may target one or more of the
following cell types: hepatocytes, endothelial, neuronal,
cardiacmyocytes, skeletal muscle cells (myoblast or myotube),
smooth muscle cells, fibroblasts, erythrocytes, T-cells, B-cells,
leukocytes, osteoblasts, chondrocytes, adult stem cells, embryonic
stem cells, and tumor cells.
[0039] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide may be a fusogenic peptide or fragment
thereof.
[0040] Lipopeptides of this disclosure can be prepared where, for
example, the central peptide may be a SNARE (solutable NSF
attachment receptor) peptide or fragment thereof. For example, the
central peptide may be a v-SNARE or t-SNARE. The central peptide
may be a NSF (N-ethylamaleimide-sensitive factor) or SNAP (soluable
NSF attachment protein).
[0041] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 1
##STR00001##
where R.sup.1, R.sup.2, R.sup.N, J, Z, R.sup.3, R.sup.4, and n are
defined as above.
[0042] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be C14alkyl, C16alkyl,
C18alkyl, or (C18:1)alkenyl.
[0043] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be lipoid groups.
[0044] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, selected lipid-like tails
which impart sufficient lipophilic character or lipophilicity, such
as defined by water/octanol partitioning, to provide delivery
across a membrane or uptake by a cell. In some embodiments,
R.sup.2, R.sup.N, R.sup.3 and R.sup.4 may independently, for each
occurrence, selected lipid-like tails which impart sufficient
lipophilic character or lipophilicity to provide cell membrane
anchoring. These tails provide, when used in an amino acid lipid
structure, an amphipathic molecule. Lipid-like tails may be derived
from phospholipids, glycolipids, triacylglycerols,
glycerophospholipids, sphingolipids, ceramides, sphingomyelins,
cerebrosides, or gangliosides, among others, and may contain a
steroid.
[0045] In certain embodiments, R.sup.2, R.sup.N, R.sup.3 and
R.sup.4 may independently, for each occurrence, be a lipid-like
tail having a glycerol backbone.
[0046] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be C3alkyl, C4alkyl,
C5alkyl, C6alkyl, C7alkyl, C8alkyl, C9alkyl, C10alkyl, C11alkyl,
C12alkyl, C13alkyl, C14alkyl, C15alkyl, C16alkyl, C17alkyl,
C18alkyl, C19alkyl, C20alkyl, C21alkyl, or C22alkyl.
[0047] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be lipophilic tails having
one of the following structures:
##STR00002##
In the structures above, X represents Z, J, N, or the alpha-carbon
of Structure I above, and is counted as one of the atoms in the
numerical designation, for example, "18:3." In some embodiments, X
may be a carbon, nitrogen, or oxygen atom.
[0048] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be lipophilic tails having
one of the following structures:
##STR00003##
where X is as defined above.
[0049] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be selected lipid-like
tails which may contain a cholesterol, a sterol, or a steroid such
as gonanes, estranes, androstanes, pregnanes, cholanes,
cholestanes, ergostanes, campestanes, poriferastanes, stigmastanes,
gorgostanes, lanostanes, cycloartanes, as well as sterol or
zoosterol derivatives of any of the foregoing, and their biological
intermediates and precursors, which may include, for example,
cholesterol, lanosterol, stigmastanol, dihydrolanosterol,
zymosterol, zymostenol, desmosterol, 7-dehydrocholesterol, and
mixtures and derivatives thereof.
[0050] In certain embodiments, R.sup.2, R.sup.N, R.sup.3 and
R.sup.4 may independently, for each occurrence, be derived from
fatty acid-like tails such as tails from myristic acid
(C14:0)alkenyl, palmitic acid (C16:0)alkenyl, stearic acid
(C18:0)alkenyl, oleic acid (C18:1, double bond at carbon 9)alkenyl,
linoleic acid (C18:2, double bond at carbon 9 or 12)alkenyl,
linonenic acid (C18:3, double bond at carbon 9, 12, or 15)alkenyl,
arachidonic acid (C20:4, double bond at carbon 5, 8, 11, or
14)alkenyl, and eicosapentaenoic acid (C20:5, double bond at carbon
5, 8, 11, 14, or 17)alkenyl. Other examples of fatty acid-like
tails are found at Donald Voet and Judith Voet, Biochemistry, 3rd
Edition (2005), p. 383.
[0051] In some embodiments, R.sup.2, R.sup.N, R.sup.3 and R.sup.4
may independently, for each occurrence, be derived from an
isoprenoid.
[0052] In some embodiments, R.sup.3 may be a lipophilic group, as
described above, and R.sup.2, R.sup.N, and R.sup.4 may be
hydrogen.
[0053] In some embodiments, R.sup.4 may be a lipophilic group, as
described above, and R.sup.2, R.sup.N, and R.sup.3 may be
hydrogen.
[0054] In some embodiments, R.sup.3 and R.sup.4 may be a lipophilic
group, as described above, and R.sup.2 and R.sup.N may be
hydrogen.
[0055] In some embodiments, R.sup.3, R.sup.4, R.sup.2 may be a
lipophilic group, as described above, and R.sup.N may be
hydrogen.
[0056] In some embodiments, R.sup.3, R.sup.4, R.sup.N may be a
lipophilic group, as described above, and R.sup.2 may be
hydrogen.
[0057] In some embodiments, R.sup.3 and R.sup.N may be a lipophilic
group, as described above, and R.sup.2 and R.sup.4 may be
hydrogen.
[0058] In some embodiments, R.sup.N and R.sup.4 may be a lipophilic
group, as described above, and R.sup.2 and R.sup.3 may be
hydrogen.
[0059] In some embodiments, R.sup.N and R.sup.2 may be a lipophilic
group, as described above, and R.sup.3 and R.sup.4 may be
hydrogen.
[0060] In some embodiments, R.sup.4 and R.sup.2 may be a lipophilic
group, as described above, and R.sup.3 and R.sup.N may be
hydrogen.
[0061] In some embodiments, R.sup.3 and R.sup.2 may be a lipophilic
group, as described above, and R.sup.4 and R.sup.N may be
hydrogen.
[0062] In some embodiments, a lipophilic group may be linked to the
alpha-carbon of any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100 sequential amino acid residues. In some embodiments, a
lipophilic group may be linked to the alpha-carbon of every other
amino acid residue of the central peptide. In some embodiments, a
lipophilic group may be linked to the alpha-carbon of any two
sequential amino acid residues of the central peptide where the two
sequential amino acid residues are each adjacent to an amino acid
residue within the central peptide that do not have a lipophilic
group linked to its alpha-carbon.
[0063] In some embodiments, a lipophilic group may be linked to the
amine group of any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100 sequential amino acid residues where the amine groups is
adjacent to the alpha-carbon of the amino acid residue of the
central peptide. In some embodiments, a lipophilic group may be
linked to the amine group of every other amino acid residue of the
central peptide. In some embodiments, a lipophilic group may be
linked to the amine group of any two sequential amino acid residues
of the central peptide where the two sequential amino acid residues
are each adjacent to an amino acid residue within the central
peptide that do not have a lipophilic group linked to its amine
group where the amine group is adjacent to the alpha-carbon of the
amino acid residue of the central peptide.
[0064] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 2, where z is from 0 to
20 (or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20), X is independently, for each occurrence, any
amino acid, R.sup.1 is defined above, and n is from 0 to 98 (or 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98).
##STR00004##
[0065] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 3, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00005##
[0066] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 4, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00006##
[0067] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 5, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00007##
[0068] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 6, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00008##
[0069] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 7, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00009##
[0070] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 8, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00010##
[0071] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 9, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00011##
[0072] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 10, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00012##
[0073] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 11, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00013##
[0074] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 12, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00014##
[0075] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 13, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00015##
[0076] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 14, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00016##
[0077] In some embodiments, a range of lipopeptides corresponding
to Formula I are represented by Structure 15, where z is from 0 to
20, X is independently, for each occurance, any amino acid, R.sup.1
is defined above, and n is from 0 to 98.
##STR00017##
[0078] As used herein, the term "amino acid" includes
naturally-occurring and non-naturally occurring amino acids.
Examples of amino acids include Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and
Val. Thus, a lipopeptide of this invention can be made from a
genetically encoded amino acid, a naturally occurring
non-genetically encoded amino acid, or a synthetic amino acid. Some
examples of amino acids include 2-aminoadipic acid, 3-aminoadipic
acid, 2,3-diaminopropionic acid, 2-aminobutyric acid,
4-aminobutyric acid, 2,3-diaminobutyric acid, 2,4-diaminobutyric
acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid,
2-aminopimelic acid, 2,2'-diaminopimelic acid, 6-aminohexanoic
acid, 6-aminocaproic acid, 2-aminoheptanoic acid, desmosine,
ornithine, citrulline, N-methylisoleucine, norleucine,
tert-leucine, phenylglycine, t-butylglycine, N-methylglycine,
sacrosine, N-ethylglycine, cyclohexylglycine,
4-oxo-cyclohexylglycine, N-ethylasparagine, cyclohexylalanine,
t-butylalanine, naphthylalanine, pyridylalanine, 3-chloroalanine,
3-benzothienylalanine, 4-halophenylalanine, 4-chlorophenylalanine,
2-fluorophenylalanine, 3-fluorophenylalanine,
4-fluorophenylalanine, penicillamine, 2-thienylalanine, methionine,
methionine sulfoxide, homoarginine, norarginine, nor-norarginine,
N-acetyllysine, N-aminophenylalanine, N-methylvaline, homocysteine,
homoserine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, 6-N-methyllysine,
norvaline, O-allyl-serine, O-allyl-threonine, alpha-aminohexanoic
acid, alpha-aminovaleric acid, and pyroglutamic acid.
[0079] As used herein, the term "amino acid" includes alpha- and
beta-amino acids.
[0080] Other amino acid residues can be found in Fasman, CRC
Practical Handbook of Biochemistry and Molecular Biology, CRC
Press, Inc. (1989).
[0081] In general, a compound may contain one or more chiral
centers. Compounds containing one or more chiral centers may
include those described as an "isomer," a "stereoisomer," a
"diastereomer," an "enantiomer," an "optical isomer," or as a
"racemic mixture." Conventions for stereochemical nomenclature, for
example the stereoisomer naming rules of Cahn, Ingold and Prelog,
as well as methods for the determination of stereochemistry and the
separation of stereoisomers are known in the art. See, for example,
Michael B. Smith and Jerry March "March's Advanced Organic
Chemistry", 5th edition, 2001. The compounds and structures of this
disclosure are meant to encompass all possible isomers,
stereoisomers, diastereomers, enantiomers, and/or optical isomers
that would be understood to exist for the specified compound or
structure, including any mixture, racemic or otherwise,
thereof.
[0082] The term "lipophilic group" or "lipoid group" or "lipid-like
tail" or "lipiphilic tail" as used herein refers to an alkyl,
cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl,
acyl, heteroaryl, heterocycle, heterocyclyl, alkanoyloxy,
alkylamino, alkylaminoalkyl, aroyl, and aralkyl group including
substituted variations thereof.
[0083] The term "alkyl" as used herein refers to a saturated,
branched or unbranched, substituted or unsubstituted aliphatic
group containing from 1-22 carbon atoms. This definition applies to
the alkyl portion of other groups such as, for example, alkoxy,
alkanoyl, aralkyl, and other groups defined below. The term
"cycloalkyl" as used herein refers to a saturated, substituted or
unsubstituted cyclic alkyl ring containing from 3 to 12 carbon
atoms. The term "alkenyl" as used herein refers to an unsaturated,
branched or unbranched, substituted or unsubstituted alkyl or
cycloalkyl having 2 to 22 carbon atoms and at least one
carbon-carbon double bond. The term "alkynyl" as used herein refers
to an unsaturated, branched or unbranched, substituted or
unsubstituted alkyl or cycloalkyl having 2 to 22 carbon atoms and
at least one carbon-carbon triple bond.
[0084] The term "alkoxy" as used herein refers to an alkyl,
cycloalkyl, alkenyl, or alkynyl group covalently bonded to an
oxygen atom. The term "alkanoyl" as used herein refers to
--C(.dbd.O)-alkyl, which may alternatively be referred to as
"acyl." The term "alkanoyloxy" as used herein refers to
--O--C(.dbd.O)-alkyl groups. The term "alkylamino" as used herein
refers to the group --NRR', where R and R' are each either hydrogen
or alkyl, and at least one of R and R' is alkyl. Alkylamino
includes groups such as piperidino wherein R and R' form a ring.
The term "alkylaminoalkyl" refers to -alkyl-NRR'.
[0085] The term "aryl" as used herein refers to any stable
monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to
12 atoms in each ring, wherein at least one ring is aromatic. Some
examples of an aryl include phenyl, naphthyl, tetrahydro-naphthyl,
indanyl, and biphenyl. Where an aryl substituent is bicyclic and
one ring is non-aromatic, it is understood that attachment is to
the aromatic ring. An aryl may be substituted or unsubstituted.
[0086] The term "heteroaryl" as used herein refers to any stable
monocyclic, bicyclic, or polycyclic carbon ring system of from 4 to
12 atoms in each ring, wherein at least one ring is aromatic and
contains from 1 to 4 heteroatoms selected from oxygen, nitrogen and
sulfur. Some examples of a heteroaryl include acridinyl,
quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl,
benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl,
isoxazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl,
pyrrolyl, and tetrahydroquinolinyl. A heteroaryl includes the
N-oxide derivative of a nitrogen-containing heteroaryl.
[0087] The term "heterocycle" or "heterocyclyl" as used herein
refers to an aromatic or nonaromatic ring system of from five to
twenty-two atoms, wherein from 1 to 4 of the ring atoms are
heteroatoms selected from oxygen, nitrogen, and sulfur. Thus, a
heterocycle may be a heteroaryl or a dihydro or tetrathydro version
thereof.
[0088] The term "aroyl" as used herein refers to an aryl radical
derived from an aromatic carboxylic acid, such as a substituted
benzoic acid. The term "aralkyl" as used herein refers to an aryl
group bonded to an alkyl group, for example, a benzyl group.
[0089] The term "carboxyl" as used herein represents a group of the
formula --C(.dbd.O)OH or --C(.dbd.O)O.sup.-. The terms "carbonyl"
and "acyl" as used herein refer to a group in which an oxygen atom
is double-bonded to a carbon atom >C.dbd.O. The term "hydroxyl"
as used herein refers to --OH or --O.sup.-. The term "nitrile" or
"cyano" as used herein refers to --CN. The term "halogen" or "halo"
refers to fluoro (--F), chloro (--Cl), bromo (--Br), and iodo
(--I).
[0090] The term "substituted" as used herein refers to an atom
having one or more substitutions or substituents which can be the
same or different and may include a hydrogen substituent. Thus, the
terms alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl,
alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl,
heterocycle, aroyl, and aralkyl as used herein refer to groups
which include substituted variations. Substituted variations
include linear, branched, and cyclic variations, and groups having
a substituent or substituents replacing one or more hydrogens
attached to any carbon atom of the group. Substituents that may be
attached to a carbon atom of the group include alkyl, cycloalkyl,
alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino,
alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl, aralkyl,
acyl, hydroxyl, cyano, halo, haloalkyl, amino, aminoacyl,
alkylaminoacyl, acyloxy, aryloxy, aryloxyalkyl, mercapto, nitro,
carbamyl, carbamoyl, and heterocycle. For example, the term ethyl
includes without limitation --CH.sub.2CH.sub.3, --CHFCH.sub.3,
--CF.sub.2CH.sub.3, --CHFCH.sub.2F, --CHFCHF.sub.2, --CHFCF.sub.3,
--CF.sub.2CH.sub.2F, --CF.sub.2CHF.sub.2, --CF.sub.2CF.sub.3, and
other variations as described above.
[0091] A pharmaceutically acceptable salt of a peptide or protein
composition of this invention which is sufficiently basic may be an
acid-addition salt with, for example, an inorganic or organic acid
such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric,
chlorosulfonic, trifluoroacetic, citric, maleic, acetic, propionic,
oxalic, malic, maleic, malonic, fumaric, or tartaric acids, and
alkane- or arenesulfonic acids such as methanesulfonic,
ethanesulfonic, benzenesulfonic, chlorobenzenesulfonic,
toluenesulfonic, naphthalenesulfonic, naphthalenedisulfonic, and
camphorsulfonic acids.
[0092] A pharmaceutically acceptable salt of a peptide or protein
composition of this invention which is sufficiently acidic may be
an alkali metal salt, for example, a sodium or potassium salt, or
an alkaline earth metal salt, for example, a calcium or magnesium
salt, or an ammonium salt or a salt with an organic base which
provides a physiologically-acceptable cation, for example, a salt
with methylamine, dimethylamine, trimethylamine, triethylamine,
ethanolamine, diethanolamine, triethanolamine, ethylenediamine,
tromethamine, N-methylglucamine, piperidine, morpholine or
tris-(2-hydroxyethyl)amine. See, for example, Berge et al., J.
Pharm. Sci. 66:1, 1971.
[0093] Some compounds, peptides and/or protein compositions of this
invention may have one or more chiral centers and/or geometric
isomeric centers (E- and Z-isomers), and it is to be understood
that the invention encompasses all such optical isomers,
diastereoisomers and geometric isomers.
[0094] This invention encompasses any and all tautomeric, solvated
or unsolvated, and hydrated or unhydrated forms of the compounds,
peptides and/or protein compositions disclosed herein.
Uses for Regulatory RNA and RNA Interference
[0095] In some aspects, this disclosure relates generally to the
fields of regulatory RNA and RNA interference, antisense
therapeutics, and delivery of RNA therapeutics. More particularly,
this invention relates to compositions and formulations for
ribonucleic acids, and their uses for medicaments and for delivery
as therapeutics. This invention relates generally to methods of
using ribonucleic acids in RNA interference for gene-specific
inhibition of gene expression in cells, or in mammals to alter a
disease state or a phenotype.
[0096] RNA interference refers to methods of sequence-specific
post-transcriptional gene silencing which is mediated by a
double-stranded RNA (dsRNA) called a short interfering RNA (siRNA).
See Fire, et al., Nature 391:806, 1998, and Hamilton, et al.,
Science 286:950-951, 1999. RNAi is shared by diverse flora and
phyla and is believed to be an evolutionarily-conserved cellular
defense mechanism against the expression of foreign genes. See
Fire, et al., Trends Genet. 15:358, 1999.
[0097] RNAi is therefore a ubiquitous, endogenous mechanism that
uses small noncoding RNAs to silence gene expression. See
Dykxhoorn, D. M. and J. Lieberman, Annu. Rev. Biomed. Eng.
8:377-402, 2006. RNAi can regulate important genes involved in cell
death, differentiation, and development. RNAi may also protect the
genome from invading genetic elements, encoded by transposons and
viruses. When a siRNA is introduced into a cell, it binds to the
endogenous RNAi machinery to disrupt the expression of mRNA
containing complementary sequences with high specificity. Any
disease-causing gene and any cell type or tissue can potentially be
targeted. This technique has been rapidly utilized for
gene-function analysis and drug-target discovery and validation.
Harnessing RNAi also holds great promise for therapy, although
introducing siRNAs into cells in vivo remains an important
obstacle.
[0098] The mechanism of RNAi, although not yet fully characterized,
is through cleavage of a target mRNA. The RNAi response involves an
endonuclease complex known as the RNA-induced silencing complex
(RISC), which mediates cleavage of a single-stranded RNA
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
(Elbashir, et al., Genes Dev. 15:188, 2001).
[0099] One way to carry out RNAi is to introduce or express a siRNA
in cells. Another way is to make use of an endogenous ribonuclease
III enzyme called dicer. One activity of dicer is to process a long
dsRNA into siRNAs. See Hamilton, et al., Science 286:950-951, 1999;
Berstein, et al., Nature 409:363, 2001. A siRNA derived from dicer
is typically about 21-23 nucleotides in overall length with about
19 base pairs duplexed. See Hamilton, et al., supra; Elbashir, et
al., Genes Dev. 15:188, 2001. In essence, a long dsRNA can be
introduced in a cell as a precursor of a siRNA.
[0100] This invention provides a range of compositions,
formulations and methods which include a regulatory RNA, an
interfering nucleic acid or a precursor thereof in combination with
various components including lipids, amino acid lipids, and natural
or synthetic polymers.
[0101] The term "dsRNA" as used herein refers to any nucleic acid
molecule comprising at least one ribonucleotide molecule and
capable of inhibiting or down regulating gene expression, for
example, by promoting RNA interference ("RNAi") or gene silencing
in a sequence-specific manner. The dsRNAs of this disclosure may be
suitable substrates for Dicer or for association with RISC to
mediate gene silencing by RNAi. One or both strands of the dsRNA
can further comprise a terminal phosphate group, such as a
5'-phosphate or 5',3'-diphosphate. As used herein, dsRNA molecules,
in addition to at least one ribonucleotide, can further include
substitutions, chemically-modified nucleotides, and
non-nucleotides. In certain embodiments, dsRNA molecules comprise
ribonucleotides up to about 100% of the nucleotide positions.
[0102] Examples of dsRNA molecules can be found in, for example,
U.S. patent application Ser. No. 11/681,725, U.S. Pat. Nos.
7,022,828 and 7,034,009, and PCT International Application
Publication No. WO/2003/070897.
[0103] In addition, as used herein, the term "dsRNA" is meant to be
synonymous with other terms used to describe nucleic acid molecules
that are capable of mediating sequence specific RNAi, for example,
meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA
(gdsRNA), short interfering nucleic acid (siNA), siRNA, micro-RNA
(miRNA), short hairpin RNA (shRNA), short interfering
oligonucleotide, short interfering substituted oligonucleotide,
short interfering modified oligonucleotide, chemically-modified
dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), among
others. The term "large double-stranded (ds) RNA" refers to any
double-stranded RNA longer than about 40 base pairs (bp) to about
100 by or more, particularly up to about 300 by to about 500 bp.
The sequence of a large dsRNA may represent a segment of an mRNA or
an entire mRNA. A double-stranded structure may be formed by
self-complementary nucleic acid molecule or by annealing of two or
more distinct complementary nucleic acid molecule strands.
[0104] In some aspects, a dsRNA comprises two separate
oligonucleotides, comprising a first strand (antisense) and a
second strand (sense), wherein the antisense and sense strands are
self-complementary (i.e., each strand comprises a nucleotide
sequence that is complementary to a nucleotide sequence in the
other strand and the two separate strands form a duplex or
double-stranded structure, for example, wherein the double-stranded
region is about 15 to about 24 base pairs or about 26 to about 40
base pairs); the antisense strand comprises a nucleotide sequence
that is complementary to a nucleotide sequence in a target nucleic
acid molecule or a portion thereof (e.g., a human mRNA); and the
sense strand comprises a nucleotide sequence corresponding (i.e.,
homologous) to the target nucleic acid sequence or a portion
thereof (e.g., a sense strand of about 15 to about 25 nucleotides
or about 26 to about 40 nucleotides corresponds to the target
nucleic acid or a portion thereof).
[0105] In some aspects, the dsRNA may be assembled from a single
oligonucleotide in which the self-complementary sense and antisense
strands of the dsRNA are linked by together by a nucleic acid
based-linker or a non-nucleic acid-based linker. In some
embodiments, the first (antisense) and second (sense) strands of
the dsRNA molecule are covalently linked by a nucleotide or
non-nucleotide linker as described herein and known in the art. In
some embodiments, a first dsRNA molecule is covalently linked to at
least one second dsRNA molecule by a nucleotide or non-nucleotide
linker known in the art, wherein the first dsRNA molecule can be
linked to a plurality of other dsRNA molecules that can be the same
or different, or any combination thereof. In some embodiments, the
linked dsRNA may include a third strand that forms a meroduplex
with the linked dsRNA.
[0106] In some respects, dsRNA molecules described herein form a
meroduplex RNA (mdRNA) having three or more strands, for example,
an `A` (first or antisense) strand, `S1` (second) strand, and `S2`
(third) strand in which the `S1` and `S2` strands are complementary
to and form base pairs (bp) with non-overlapping regions of the `A`
strand (e.g., an mdRNA can have the form of A:S1S2). The S1, S2, or
more strands together essentially comprise a sense strand to the
`A` strand. The double-stranded region formed by the annealing of
the `S1` and `A` strands is distinct from and non-overlapping with
the double-stranded region formed by the annealing of the `S2` and
`A` strands. An mdRNA molecule is a "gapped" molecule, meaning a
"gap" ranging from 0 nucleotides up to about 10 nucleotides. In
some embodiments, the A:S1 duplex is separated from the A:S2 duplex
by a gap resulting from at least one unpaired nucleotide (up to
about 10 unpaired nucleotides) in the `A` strand that is positioned
between the A:S1 duplex and the A:S2 duplex and that is distinct
from any one or more unpaired nucleotide at the 3'-end of one or
more of the `A`, `S1`, or `S2` strands. In some embodiments, the
A:S1 duplex is separated from the A:B2 duplex by a gap of zero
nucleotides (i.e., a nick in which only a phosphodiester bond
between two nucleotides is broken or missing in the polynucleotide
molecule) between the A:S1 duplex and the A: S2 duplex--which can
also be referred to as nicked dsRNA (ndsRNA). For example, A:S1S2
may be comprised of a dsRNA having at least two double-stranded
regions that combined total about 14 base pairs to about 40 base
pairs and the double-stranded regions are separated by a gap of
about 0 to about 10 nucleotides, optionally having blunt ends, or
A:S1S2 may comprise a dsRNA having at least two double-stranded
regions separated by a gap of up to 10 nucleotides wherein at least
one of the double-stranded regions comprises between about 5 base
pairs and 13 base pairs.
[0107] As described herein, a dsRNA molecule which contains three
or more strands may be referred to as a "meroduplex" RNA (mdRNA).
Examples of mdRNA molecules can be found in U.S. Provisional Patent
Application Nos. 60/934,930 and 60/973,398.
[0108] A dsRNA or large dsRNA may include a substitution or
modification in which the substitution or modification may be in a
phosphate backbone bond, a sugar, a base, or a nucleoside. Such
nucleoside substitutions can include natural non-standard
nucleosides (e.g., 5-methyluridine or 5-methylcytidine or a
2-thioribothymidine), and such backbone, sugar, or nucleoside
modifications can include an alkyl or heteroatom substitution or
addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or
sulfur, or other modifications known in the art.
[0109] The term "pyrimidine" as used herein refers to conventional
pyrimidine bases, including standard pyrimidine bases uracil and
cytosine. In addition, the term pyrimidine is contemplated to
embrace natural non-standard pyrimidine bases or acids, such as
5-methyluracil, 4-thiouracil, pseudouracil, dihydrouracil, orotate,
5-methylcytosine, or the like, as well as a chemically-modified
bases or "universal bases," which can be used to substitute for a
standard pyrimidine within nucleic acid molecules of this
disclosure. Examples of pyrmidines suitable for use within a dsRNA
of this disclosure include those disclosed in U.S. Pat. No.
6,846,827, hereby incorporated by reference.
[0110] The term "purine" as used herein refers to conventional
purine bases, including standard purine bases adenine and guanine.
In addition, the term purine is contemplated to embrace natural
non-standard purine bases or acids, such as N2-methylguanine,
inosine, 2,6-aminopurine, or the like, as well as a
chemically-modified bases or "universal bases," which can be used
to substitute for a standard purine within nucleic acid molecules
of this disclosure.
[0111] In yet another embodiment, the mdRNA or dsRNA comprises one
or more nucleotides having the formula:
##STR00018##
wherein, X is O or CH.sub.2, Y is O, and Z is CH.sub.2; R.sub.1 is
selected from the group consisting of adenine, cytosine, guanine,
hypoxanthine, uracil, thymine, 2,6-diaminopurine, C-phenyl,
C-naphthyl, inosine, azole carboxamide,
1-.beta.-D-ribofuranosyl-4-nitroindole,
1-.beta.-D-ribofuranosyl-5-nitroindole,
1-.beta.-D-ribofuranosyl-6-nitroindole, or
1-.beta.-D-ribofuranosyl-3-nitropyrrole, and a heterocycle wherein
the heterocycle is selected from the group consisting of a
substituted 1,3-diazine, unsubstituted 1,3-diazine, and an
unsubstituted 7H imidazo[4,5]1,3 diazine; and R.sub.2, R.sub.3 are
independently selected from a group consisting of H, OH, DMTO,
TBDMSO, BnO, THPO, AcO, BzO, OP(NiPr.sub.2)O(CH.sub.2).sub.2CN,
OPO.sub.3H, diphosphate, and triphosphate, wherein R.sub.2 and
R.sub.3 together may be PhCHO.sub.2, TIPDSO.sub.2 or
DTBSO.sub.2.
[0112] In yet another embodiment, the mdRNA or dsRNA comprises one
or more are universal-binding nucleotide. Non-limiting examples of
universal-binding nucleotide include C-phenyl, C-naphthyl, inosine,
azole carboxamide, 1-.beta.-D-ribofuranosyl-4-nitroindole,
1-.beta.-D-ribofuranosyl-5-nitroindole,
1-.beta.-D-ribofuranosyl-6-nitroindole, or
1-.beta.-D-ribofuranosyl-3-nitropyrrole. Within certain aspects,
the present disclosure provides methods of using mdRNA or dsRNA
that decreases expression of a target gene by RNAi, and
compositions comprising one or more mdRNA or dsRNA, wherein at
least one mdRNA or dsRNA comprises one or more universal-binding
nucleotide(s) in the first, second or third position in the
anti-codon of the antisense strand of the mdRNA or dsRNA duplex and
wherein the mdRNA or dsRNA is capable of specifically binding to a
target sequence, such as an RNA expressed by a cell. In cases
wherein the sequence of the target RNA includes one or more single
nucleotide substitutions, mdRNA or dsRNA comprising a
universal-binding nucleotide retains its capacity to specifically
bind a target RNA, thereby mediating gene silencing and, as a
consequence, overcoming escape of the target from mdRNA or
dsRNA-mediated gene silencing.
[0113] Non-limiting examples for the above compositions includes
modifying the anti-codons for tyrosine (AUA) or phenylalanine (AAA
or GAA), cysteine (ACA or GCA), histidine (AUG or GUG), asparagine
(AUU or GUU), isoleucine (UAU) and aspartate (AUC or GUC) within
the anti-codon of the antisense strand of the mdRNA or dsRNA
molecule.
[0114] For example, within certain embodiments, the isoleucine
anti-codon UAU, for which AUA is the cognate codon, may be modified
such that the third-position uridine (U) nucleotide is substituted
with the universal-binding nucleotide inosine (I) to create the
anti-codon UAI. Inosine is an exemplary universal-binding
nucleotide that can nucleotide-pair with an adenosine (A), uridine
(U), and cytidine (C) nucleotide, but not guanosine (G). This
modified anti-codon UAI increases the specific-binding capacity of
the mdRNA or dsRNA molecule and thus permits the mdRNA or dsRNA to
pair with mRNAs having any one of AUA, UUA, and CUA in the
corresponding position of the coding strand thereby expanding the
number of available RNA degradation targets to which the mdRNA or
dsRNA may specifically bind.
[0115] Alternatively, the anti-codon AUA may also or alternatively
be modified by substituting a universal-binding nucleotide in the
third or second position of the anti-codon such that the
anti-codon(s) represented by UAI (third position substitution) or
UIU (second position substitution) to generate mdRNA or dsRNA that
are capable of specifically binding to AUA, CUA and UUA and AAA,
ACA and AUA.
[0116] In certain aspects, mdRNA or dsRNA disclosed herein can
include between about 1 universal-binding nucleotide and about 10
universal-binding nucleotides. Within certain aspects, the
presently disclosed mdRNA or dsRNA may comprise a sense strand that
is homologous to a sequence of a target gene and an antisense
strand that is complementary to the sense strand, with the proviso
that at least one nucleotide of the antisense strand of the
otherwise complementary mdRNA or dsRNA duplex is replaced by one or
more universal-binding nucleotide.
[0117] It will be understood that, regardless of the position at
which the one or more universal-binding nucleotide is substituted,
the mdRNA or dsRNA molecule is capable of binding to a target gene
and one or more variant(s) thereof thereby facilitating the
degradation of the target gene or variant thereof via Dicer or a
RISC complex. Thus, the mdRNA or dsRNA of the present disclosure
are suitable for introduction into cells to mediate targeted
post-transcriptional gene silencing of a TNF gene or variants
thereof. When a mdRNA or dsRNA is inserted into a cell, the mdRNA
or dsRNA duplex is then unwound, and the antisense strand anneals
with mRNA to form a Dicer substrate or the antisense strand is
loaded into an assembly of proteins to form the RNA-induced
silencing complex (RISC).
[0118] In yet another embodiment, the mdRNA or dsRNA comprises one
or more have a 2'-sugar substitution. Non-limiting examples of a
2'-sugar substitution include 2'-O-methyl, 2'-O-methoxyethyl,
2'-O-2-methoxyethyl, wherein the 2'-sugar substitution is a
halogen, or wherein the 2'-sugar substitution is a 2'-fluoro, or
wherein the 2'-sugar substitution is a 2'-O-allyl.
[0119] In yet another embodiment, the mdRNA or dsRNA comprising at
least one, two, three, four, five, or more base pairs (including 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base
pairs), each base pair comprising a 5-methyluridine base paired
with a 2,6-diaminopurine, and wherein the 5-methyluridine of each
base pair is in the guide strand (antisense strand) or wherein the
5-methyluridine of each base pair is in the passenger strand (sense
strand).
[0120] In any one embodiment of the disclosure, a base pair
comprising a 5-methyluridine base paired with a 2,6-diaminopurine
in a double-stranded region of the RNA may be as follows:
##STR00019##
In this schematic, the 5-methyluridne:2,6-diaminopurine base pair
has three hydrogen bonds (a hashed line represents a hydrogen
bond). The base pair may have 1, 2 or 3 hydrogen bonds, preferably
the base pair has 1 hydrogen bond, more preferably two hydrogen
bonds and most preferably three hydrogen bonds.
[0121] In anyone embodiment of the disclosure, the double-stranded
region of an RNA comprises at least one non-standard base pair
comprising:
##STR00020##
In this schematic, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8
are independently any one or more organic group consisting of one
to twenty (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20) atoms selected from carbon, oxygen, nitrogen,
sulfur, hydrogen, selenium, silicon, halogen, chlorine, fluorine,
and bromine. A dashed line indicates an optional bond that is
either present or absent within the structures above. In this
schematic, hydrogen bonds are not indicated in the above
structures; however, such hydrogen bonds would form between the
hydrogen bond donor and hydrogen bond acceptor groups of R.sub.4
and R.sub.7, between the hydrogen bond donor group of R.sub.5 and
the nitrogen (a hydrogen bond acceptor) in the third position of
the pyrimidine structure above, and between the hydrogen bond donor
and hydrogen bond acceptor group of R.sub.6 and R.sub.9. In an
embodiment of this disclosure, R4 has a hydrogen bond donor group
and R7 has a hydrogen bond acceptor group, R5 has a hydrogen bond
donor group, and R6 has a hydrogen bond donor group and R9 has an
hydrogen bond acceptor group. In another embodiment, R4 has an
hydrogen bond acceptor group and R7 has a hydrogen bond donor
group, R5 has a hydrogen bond donor group, and R6 has a hydrogen
bond donor group and R9 has an hydrogen bond acceptor group. In
another embodiment, R4 has an hydrogen bond acceptor group and R7
has a hydrogen bond donor group, R5 has a hydrogen bond donor
group, and R6 has an hydrogen bond acceptor group and R9 has a
hydrogen bond donor group.
[0122] In anyone embodiment of the disclosure, the double-stranded
region of an RNA comprises at least one non-standard base pair
comprising:
##STR00021##
In this schematic, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, and
R.sub.9 are independently any one or more organic group consisting
of one to twenty (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20) atoms selected from carbon, oxygen,
nitrogen, sulfur, hydrogen, selenium, silicon, halogen, chlorine,
fluorine, and bromine A dashed line indicates an optional bond that
is either present or absent within the structures above. A hashed
line between the substituent groups of the two structures above
indicates the presence of a hydrogen bond (three hydrogen bonds are
shown in this schematic). In another embodiment, R4 has a donor
group and R7 has an acceptor group. In another embodiment, R4 has
an acceptor group and R7 has an acceptor group. In another
embodiment, R5 has a donor group and R8 has an acceptor group. In
another embodiment, R5 has an acceptor group and R8 has an acceptor
group. In another embodiment, R6 has a donor group and R9 has an
acceptor group. In another embodiment, R6 has an acceptor group and
R9 has an acceptor group.
[0123] Another aspect of the disclosure is a mdRNA or dsRNA
comprising an acyclic nucleotide monomer. In a preferred
embodiment, the acyclic nucleotide monomer is a
2'-3'-seco-nucleotide monomer. Preferably, the acyclic nucleotide
monomer is selected from the group consisting of monomer E, F, G,
H, I or J (see below).
##STR00022## ##STR00023##
Other examples of acyclic nucleotide monomers are described, for
example, in PCT patent application PCT/US2008/64417, hereby
incorporated by reference in their entirety.
[0124] In addition, as used herein, the term "RNAi" is meant to be
equivalent to other terms used to describe sequence specific RNA
interference, such as post transcriptional gene silencing,
translational inhibition, or epigenetics. For example, dsRNA
molecules of this disclosure can be used to epigenetically silence
genes at the post-transcriptional level or the pre-transcriptional
level or any combination thereof.
[0125] In some aspects, this invention provides compositions
containing one or more RNAi-inducing agents which are targeted to
one or more target transcripts, along with one or more delivery
components. Examples of delivery components include lipopeptides,
lipids, peptides with attached lipoid moieties or attached natural
or synthetic polymers, and polymeric lipids.
[0126] The compositions and formulations of this invention may be
used for delivery of RNAi-inducing entities such as dsRNA, siRNA,
mdRNA, miRNA, shRNA, or RNAi-inducing vectors to cells in intact
mammalian subjects, and may also be used for delivery of these
agents to cells in culture.
[0127] This invention also provides methods for the delivery of one
or more RNAi-inducing entities to organs and tissues within the
body of a mammal. In some embodiments, compositions containing an
RNAi-inducing entity and one or more lipopeptide components are
introduced by various routes to be transported within the body and
taken up by cells in one or more organs or tissues, where
expression of a target transcript is modulated.
[0128] This invention provides pharmaceutically-acceptable nucleic
acid compositions with various lipopeptides, lipids, or peptides
having attached lipoid moieties or natural or synthetic polymers
which are useful for therapeutic delivery of nucleic acids and
gene-silencing RNAs. In particular, this invention provides
compositions and methods for in vitro and in vivo delivery of
dsRNAs for decreasing, downregulating, or silencing the translation
of a target nucleic acid sequence or expression of a gene. These
compositions and methods may be used for prevention and/or
treatment of diseases in a mammal
[0129] In exemplary methods of this invention, a ribonucleic acid
molecule such as a dsRNA, siRNA, mdRNA, or shRNA is contacted with
a lipopeptide to formulate a composition which can be administered
to cells or subjects such as mammals. In some embodiments, this
invention provides methods for delivering an interfering-RNA agent
such as a dsRNA, siRNA, mdRNA, or shRNA intracellularly by
contacting a nucleic acid-containing composition with a cell.
[0130] In exemplary embodiments, this invention includes
compositions containing a nucleic acid molecule, such as a
double-stranded ribonucleic acid (dsRNA), a short interfering RNA
(siRNA), a meroduplex RNA (mdRNA), or a short hairpin RNA (shRNA),
admixed or complexed with a lipopeptide to form a composition that
enhances intracellular delivery of the nucleic acid molecule. In
some embodiments, a delivery composition of this invention may
contain an interfering-RNA agent and one, two, or more
lipopeptides, as well as one or more polymeric lipids.
[0131] In certain embodiments, the N/P ratio of the lipopeptide and
nucleic acid is from about 0.5 to about 8, from about 1 to about 4,
or about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,
2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9 or about 4.
[0132] The compositions of this invention can form stable particles
which may incorporate an interfering RNA agent. Compositions and
formulations of this invention may include further
delivery-enhancing components or excipients.
[0133] In some embodiments, compositions of this invention contain
stable RNA-lipid particles having diameters from about 5 nm to
about 400 nm. In some embodiments, the particles may have a uniform
diameter of from about 10 nm to about 300 nm. In some embodiments,
the particles may have a uniform diameter of from about 50 nm to
about 150 nm.
[0134] Within exemplary compositions of this invention, an
interfering-RNA agent may be admixed or complexed with lipopeptides
to form a composition that enhances intracellular delivery of the
dsRNA as compared to contacting target cells with naked dsRNA.
Lipids for RNA Delivery and Administration
[0135] In some aspects of this invention, lipopeptides and
additional lipids are employed for delivery and administration of
RNA components. More particularly, a composition of this invention
may include one or more lipopeptides, which may be cationic, along
with other cationic lipids and non-cationic lipids.
[0136] Cationic lipids may be monocationic or polycationic.
Non-cationic lipids include neutral lipids and lipids having
approximately zero net charge at a particular pH, for example, a
zwitterionic lipid. Non-cationic lipids also include anionic
lipids.
[0137] In some embodiments, a composition is a mixture or complex
of an RNA component with a lipopeptide and a (non-lipopeptide)
cationic lipid. In some embodiments, a composition may be a mixture
or complex of one or more interfering RNA agents with one or more
lipopeptides and one or more cationic lipids.
[0138] Examples of cationic lipids include
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane (DOTAP),
1,2-bis(dimyrstoyloxy)-3-3-(trimethylammonia)propane (DMTAP);
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide
(DMRIE); dimethyldioctadecylammonium bromide (DDAB);
3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol);
3.beta.-[N',N'-diguanidinoethyl-aminoethane)carbamoyl cholesterol
(BGTC);
2-(2-(3-(bis(3-aminopropyl)amino)propylamino)acetamido)-N,N-ditetradecyla-
cetamide (RPR209120); pharmaceutically acceptable salts thereof,
and mixtures thereof.
[0139] Examples of cationic lipids include
1,2-dialkenoyl-sn-glycero-3-ethylphosphocholines (EPCs), such as
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine,
1,2-distearoyl-sn-glycero-3-ethylphosphocholine,
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, pharmaceutically
acceptable salts thereof, and mixtures thereof.
[0140] Examples of polycationic lipids include
tetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleyl
spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS),
tetramethyltetramyristyl spermine (TMTMS), tetramethyldioleyl
spermine (TMDOS), pharmaceutically acceptable salts thereof, and
mixtures thereof.
[0141] Examples of polycationic lipids include
2,5-bis(3-aminopropylamino)-N-(2-(dioctadecylamino)-2-oxoethyl)
pentanamide (DOGS);
2,5-bis(3-aminopropylamino)-N-(2-(di(Z)-octadeca-9-dienylamino)-2-oxoethy-
l) pentanamide (DOGS-9-en);
2,5-bis(3-aminopropylamino)-N-(2-(di(9Z,12Z)-octadeca-9,12-dienylamino)-2-
-oxoethyl) pentanamide (DLinGS);
3-.beta.-(N.sup.4-(N.sup.1,N.sup.8-dicarbobenzoxyspermidine)carbamoyl)cho-
lesterol (GL-67);
(9Z,9'Z)-2-(2,5-bis(3-aminopropylamino)pentanamido)propane-1,3-diyl-dioct-
adec-9-enoate (DOSPER);
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoro-acetate (DOSPA); pharmaceutically acceptable salts
thereof, and mixtures thereof.
[0142] Examples of cationic lipids include those shown in Table
3.
TABLE-US-00003 TABLE 3 Examples of Cationic Lipids Compound Name FA
Chains M.W. CAS registry # DS404-28 BGTC Cholesterol 642.96
182056-06-0 DOSPER C18:1 848.34 178532-92-8 GL-67 Cholesterol
615.00 179075-30-0 RPR209120 Myristoyl C14 695.16 433292-13-8 DOGS
C18:0 807.37 12050-77-7 DOGS (9-en) C18:1 803.34 DLinGS C18:2
799.31 DOTMA C18:1 712.57 104162-48-3
[0143] Examples of cationic lipids are described in U.S. Pat. Nos.
4,897,355; 5,279,833; 6,733,777; 6,376,248; 5,736,392; 5,334,761;
5,459,127; 5,208,036; 5,264,618; 5,283,185; 5,753,613; 5,785,992;
and U.S. Patent Publication No. 2005/0064595.
[0144] In some embodiments, the composition is a mixture or complex
of an RNA component with a lipopeptide and a non-cationic lipid. In
some embodiments, the composition is a mixture or complex of one or
more RNA components with one or more lipopeptides and one or more
non-cationic lipids. Non-cationic lipids include neutral,
zwitterionic, and anionic lipids.
[0145] In some embodiments, a composition is a mixture or complex
of an RNA component with a cationic lipopeptide and a
(non-lipopeptide) non-cationic or neutral lipid.
[0146] In some embodiments, a composition is a mixture or complex
of one or more RNA components with one or more cationic
lipopeptides, one or more (non-lipopeptide) cationic lipids, and
one or more (non-lipopeptide) non-cationic or neutral lipids.
[0147] Examples of non-cationic lipids include
1,2-Dilauroyl-sn-glycerol (DLG); 1,2-Dimyristoyl-sn-glycerol (DMG);
1,2-Dipalmitoyl-sn-glycerol (DPG); 1,2-Distearoyl-sn-glycerol
(DSG); 1,2-Dilauroyl-sn-glycero-3-phosphatidic acid (sodium salt;
DLPA); 1,2-Dimyristoyl-sn-glycero-3-phosphatidic acid (sodium salt;
DMPA); 1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid (sodium salt;
DPPA); 1,2-Distearoyl-sn-glycero-3-phosphatidic acid (sodium salt;
DSPA); 1,2-Diarachidoyl-sn-glycero-3-phosphocholine (DAPC);
1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC);
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-Dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (chloride or
triflate; DPePC); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);
1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);
1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE);
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);
1,2-Dilauroyl-sn-glycero-3-phosphoglycerol (sodium salt; DLPG);
1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt; DMPG);
1,2-Dimyristoyl-sn-glycero-3-phospho-sn-1-glycerol (ammonium salt;
DMP-sn-1-G); 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (sodium
salt; DPPG); 1,2-Distearoyl-sn-glycero-3-phosphoglycero (sodium
salt; DSPG); 1,2-Distearoyl-sn-glycero-3-phospho-sn-1-glycerol
(sodium salt; DSP-sn-1-G);
1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt; DPPS);
1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinoPC);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt;
POPG); 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium
salt; POPG); 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(ammonium salt; POPG);
1-Palmitoyl-2-4-o-sn-glycero-3-phosphocholine (P-lyso-PC);
1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-lyso-PC); and
mixtures thereof.
[0148] Examples of non-cationic lipids include polymeric compounds
and polymer-lipid conjugates or polymeric lipids, such as pegylated
lipids having PEG regions of 300, 500, 1000, 1500, 2000, 3500,
5000, or 10,000 molecular weight, including polyethyleneglycols,
N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-dimyristoyl-sn-glycero-3--
phosphoethanolamine (sodium salt; DMPE-MPEG-2000);
N-(Carbonyl-methoxypolyethyleneglycol-5000)-1,2-dimyristoyl-sn-glycero-3--
phosphoethanolamine (sodium salt; DMPE-MPEG-5000);
N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium
salt; DPPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol
5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium
salt; DPPE-MPEG-5000); N-(Carbonyl-methoxypolyethyleneglycol
750)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;
DSPE-MPEG-750); N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;
DSPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol
5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;
DSPE-MPEG-5000); sodium cholesteryl sulfate (SCS); pharmaceutically
acceptable salts thereof, and mixtures thereof.
[0149] Examples of non-cationic lipids include polymeric lipids
such as DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG, and
diacylglycerol-PEG-2000, -5000 or -10,000.
[0150] Examples of non-cationic lipids include polymeric lipids
such as multi-branched pegylated compounds, for example DSPE-PTE020
and DSPE-AM0530K.
[0151] Examples of non-cationic lipids include polymeric lipids
such as DSPE-PG8G polyglycerine lipids.
[0152] Examples of non-cationic lipids include
dioleoylphosphatidylethanolamine (DOPE),
didecanoylphosphatidylcholine (DDPC),
diphytanoylphosphatidylethanolamine (DPhPE),
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), and
1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhPC).
[0153] Examples of non-cationic lipids include cholesterols,
sterols, and steroids such as gonanes, estranes, androstanes,
pregnanes, cholanes, cholestanes, ergostanes, campestanes,
poriferastanes, stigmastanes, gorgostanes, lanostanes,
cycloartanes, as well as sterol or zoosterol derivatives of any of
the foregoing, and their biological intermediates and precursors,
which may include, for example, cholesterol, lanosterol,
stigmastanol, dihydrolanosterol, zymosterol, zymostenol,
desmosterol, 7-dehydrocholesterol, and mixtures and derivatives
thereof.
[0154] Examples of non-cationic lipids include pegylated
cholesterols, and cholestane 3-oxo(C1-22acyl) derivatives such as
cholesteryl acetate, cholesteryl arachidonate, cholesteryl
butyrate, cholesteryl hexanoate, cholesteryl caprylate, cholesteryl
n-decanoate, cholesteryl dodecanoate, cholesteryl myristate,
cholesteryl palmitate, cholesteryl behenate, cholesteryl stearate,
cholesteryl nervonate, cholesteryl pelargonate, cholesteryl
n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl
erucate, cholesteryl heptanoate, cholesteryl linolelaidate,
cholesteryl linoleate, and mixtures and derivatives thereof.
[0155] Examples of non-cationic lipids include compounds derived
from plant sterols including phytosterols, beta-sitosterol,
campesterol, ergosterol, brassicasterol, delta-7-stigmasterol,
delta-7-avenasterol, and mixtures and derivatives thereof.
[0156] Examples of non-cationic lipids include bile acids, cholic
acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid,
deoxycholic acid, lithocholic acid, methyl-lithocholic acid, and
mixtures and derivatives thereof.
[0157] Examples of non-cationic lipids include compounds derived
from steroids including glucocorticoids, cortisol, hydrocortisone,
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, and mixtures and derivatives thereof.
[0158] Examples of non-cationic lipids include compounds derived
from steroids including androgens, testosterone,
dihydrotestosterone, androstenediol, androstenedione,
androstenedione, 3.alpha.,5.alpha.-androstanediol, and mixtures and
derivatives thereof.
[0159] Examples of non-cationic lipids include compounds derived
from steroids including estrogens, estriols, estrones, estradiols,
and mixtures and derivatives thereof.
[0160] Examples of non-cationic lipids include compounds derived
from lumisterol and vitamin D compounds.
[0161] Examples of non-cationic lipids include lipids ranging from
C10:0 to C22:6 phosphoethanolamine as shown in Table 4.
TABLE-US-00004 TABLE 4 Examples of Non-cationic Lipids Name FA
chains M.W. CAS Registry # DDPE C10:0 523.64 253685-27-7 DLPE C12:0
579.76 59752-57-7 DSPE C18:0 748.08 1069-79-0 DOPE C18:1 744.05
4004-05-1 DLinPE C18:2 740.01 20707-71-5 DLenPE C18:3 735.98
34813-40-6 DARAPE C20:4 788.06 5634-86-6 DDHAPE C22:6 836.10
123284-81-1 DPhPE 16:0[(CH3)4] 804.19 201036-16-0
[0162] Examples of anionic lipids include phosphatidylserine,
phosphatidic acid, phosphatidylcholine, platelet-activation factor
(PAF), phosphatidylethanolamine, phosphatidyl-DL-glycerol,
phosphatidylinositol, phosphatidylinositol (pi(4)p, pi(4,5)p2),
cardiolipin (sodium salt), lysophosphatides, hydrogenated
phospholipids, sphingoplipids, gangliosides, phytosphingosine,
sphinganines, pharmaceutically acceptable salts thereof, and
mixtures thereof.
[0163] In certain aspects, the lipopeptide is about from 10% to
about 100% mole percentage of delivery components, not including
the nucleic acid (e.g., RNA). In other embodiments, the lipopeptide
is from about 20% to about 80%, or from about 30% to about 70%, or
from about 40% to about 60% mole percentage of delivery components,
or about 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, or 95% mole percentage of delivery components.
Additional Delivery Lipids
[0164] In some aspects of this invention, amino acid lipids and
additional non-amino acid lipids may be employed for delivery and
administration of regulatory RNA components, RNA antagonists,
interfering RNA, or nucleic acids. More particularly, a composition
of this invention may include one or more amino acid lipids along
with non-amino acid cationic lipids and non-amino acid non-cationic
lipids.
[0165] Non-amino acid cationic lipids may be monocationic or
polycationic. Some non-amino acid cationic lipids include neutral
lipids and lipids having approximately zero net charge at a
particular pH, for example, a zwitterionic lipid. Non-amino acid
non-cationic lipids also include anionic lipids.
[0166] In some embodiments, a composition is a mixture or complex
of an RNA component with an amino acid lipid and a non-amino acid
cationic lipid. In some embodiments, a composition may be a mixture
or complex of one or more regulatory or interfering RNA agents with
one or more amino acid lipids and one or more non-amino acid
cationic lipids.
[0167] The compounds and compositions of this disclosure can be
admixed with, or attached to various targeting ligands or agents to
deliver an active agent to a cell, tissue, organ or region of an
organism. Examples of targeting agents include antibodies, ligands
for receptors, peptides, proteins, lectins, (poly)saccharides,
galactose, mannose, cyclodextrins, nucleic acids, DNA, RNA,
aptamers, and polyamino acids.
[0168] Examples of non-amino acid cationic lipids include
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane (DOTAP),
1,2-bis(dimyrstoyloxy)-3-3-(trimethylammonia)propane (DMTAP);
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide
(DMRIE); dimethyldioctadecylammonium bromide (DDAB);
3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol);
3.beta.-[N',N'-diguanidinoethyl-aminoethane)carbamoyl cholesterol
(BGTC);
2-(2-(3-(bis(3-aminopropyl)amino)propylamino)acetamido)-N,N-ditetradecyla-
cetamide (RPR209120); pharmaceutically acceptable salts thereof,
and mixtures thereof.
[0169] Examples of non-amino acid cationic lipids include
1,2-dialkenoyl-sn-glycero-3-ethylphosphocholines (EPCs), such as
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine,
1,2-distearoyl-sn-glycero-3-ethylphosphocholine,
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, pharmaceutically
acceptable salts thereof, and mixtures thereof.
[0170] Examples of non-amino acid cationic lipids include
1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA),
1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA),
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and
1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA).
[0171] Examples of non-amino acid polycationic lipids include
tetramethyltetrapalmitoyl spermine (TMTPS), tetramethyltetraoleyl
spermine (TMTOS), tetramethlytetralauryl spermine (TMTLS),
tetramethyltetramyristyl spermine (TMTMS), tetramethyldioleyl
spermine (TMDOS), pharmaceutically acceptable salts thereof, and
mixtures thereof.
[0172] Examples of non-amino acid polycationic lipids include
2,5-bis(3-aminopropylamino)-N-(2-(dioctadecylamino)-2-oxoethyl)
pentanamide (DOGS);
2,5-bis(3-aminopropylamino)-N-(2-(di(Z)-octadeca-9-dienylamino)-2-oxoethy-
l) pentanamide (DOGS-9-en);
2,5-bis(3-aminopropylamino)-N-(2-(di(9Z,12Z)-octadeca-9,12-dienylamino)-2-
-oxoethyl) pentanamide (DLinGS);
3-beta-(N.sup.4-(N.sup.1,N.sup.8-dicarbobenzoxyspermidine)carbamoyl)chole-
sterol (GL-67);
(9Z,9'Z)-2-(2,5-bis(3-aminopropylamino)pentanamido)propane-1,3-diyl-dioct-
adec-9-enoate (DOSPER);
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoro-acetate (DOSPA); pharmaceutically acceptable salts
thereof, and mixtures thereof.
[0173] Examples of non-amino acid cationic lipids include DS404-28
BGTC (CAS 182056-06-0), DOSPER (CAS 178532-92-8), GL-67
(179075-30-0), RPR209120 (CAS 433292-13-8), DOGS (12050-77-7), DOGS
(9-en, C18:1), DLinGS (C18:2), and DOTMA (104162-48-3).
[0174] Examples of non-amino acid cationic lipids are described in
U.S. Pat. Nos. 4,897,355; 5,279,833; 6,733,777; 6,376,248;
5,736,392; 5,334,761; 5,459,127; 2005/0064595; 5,208,036;
5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992.
[0175] In some embodiments, the composition is a mixture or complex
of an RNA component with an amino acid lipid and a non-amino acid
non-cationic lipid. In some embodiments, the composition is a
mixture or complex of one or more RNA components with one or more
amino acid lipids and one or more non-amino acid non-cationic
lipids.
[0176] Non-amino acid non-cationic lipids include neutral,
zwitterionic, and anionic lipids. Thus, a non-cationic zwitterionic
lipid may contain a cationic head group.
[0177] Examples of non-amino acid non-cationic lipids include
1,2-Dilauroyl-sn-glycerol (DLG); 1,2-Dimyristoyl-sn-glycerol (DMG);
1,2-Dipalmitoyl-sn-glycerol (DPG); 1,2-Distearoyl-sn-glycerol
(DSG); 1,2-Dilauroyl-sn-glycero-3-phosphatidic acid (sodium salt;
DLPA); 1,2-Dimyristoyl-sn-glycero-3-phosphatidic acid (sodium salt;
DMPA); 1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid (sodium salt;
DPPA); 1,2-Distearoyl-sn-glycero-3-phosphatidic acid (sodium salt;
DSPA); 1,2-Diarachidoyl-sn-glycero-3-phosphocholine (DAPC);
1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC);
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-Dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (chloride or
triflate; DPePC); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC); 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);
1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);
1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE);
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);
1,2-Dilauroyl-sn-glycero-3-phosphoglycerol (sodium salt; DLPG);
1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (sodium salt; DMPG);
1,2-Dimyristoyl-sn-glycero-3-phospho-sn-1-glycerol (ammonium salt;
DMP-sn-1-G); 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (sodium
salt; DPPG); 1,2-Distearoyl-sn-glycero-3-phosphoglycero (sodium
salt; DSPG); 1,2-Distearoyl-sn-glycero-3-phospho-sn-1-glycerol
(sodium salt; DSP-sn-1-G);
1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt; DPPS);
1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinoPC);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium salt;
POPG); 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (sodium
salt; POPG); 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
(ammonium salt; POPG);
1-Palmitoyl-2-4-o-sn-glycero-3-phosphocholine (P-lyso-PC);
1-Stearoyl-2-lyso-sn-glycero-3-phosphocholine (S-lyso-PC); and
mixtures thereof.
[0178] Examples of non-amino acid non-cationic lipids include
polymeric compounds and polymer-lipid conjugates or polymeric
lipids, such as pegylated lipids having PEG regions of 300, 500,
1000, 1500, 2000, 3500, or 5000 molecular weight, including
polyethyleneglycols,
N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-dimyristoyl-sn-glycero-3--
phosphoethanolamine (sodium salt; DMPE-MPEG-2000);
N-(Carbonyl-methoxypolyethyleneglycol-5000)-1,2-dimyristoyl-sn-glycero-3--
phosphoethanolamine (sodium salt; DMPE-MPEG-5000);
N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium
salt; DPPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol
5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (sodium
salt; DPPE-MPEG-5000); N-(Carbonyl-methoxypolyethyleneglycol
750)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;
DSPE-MPEG-750); N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;
DSPE-MPEG-2000); N-(Carbonyl-methoxypolyethyleneglycol
5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (sodium salt;
DSPE-MPEG-5000); sodium cholesteryl sulfate (SCS); pharmaceutically
acceptable salts thereof, and mixtures thereof.
[0179] Examples of non-amino acid non-cationic lipids include
polymeric lipids such as DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG,
and diacylglycerol-PEG-2000 or -5000.
[0180] Examples of non-amino acid non-cationic lipids include
polymeric lipids such as multi-branched pegylated compounds, for
example DSPE-PTE020 and DSPE-AM0530K.
[0181] Examples of non-amino acid non-cationic lipids include
polymeric lipids such as DSPE-PG8G polyglycerine lipids.
[0182] Examples of non-amino acid non-cationic lipids include
dioleoylphosphatidylethanolamine (DOPE),
diphytanoylphosphatidylethanolamine
(DPhPE),1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), and
1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine (DPhPC).
[0183] Examples of non-amino acid non-cationic lipids include
cholesterols, sterols, and steroids such as gonanes, estranes,
androstanes, pregnanes, cholanes, cholestanes, ergostanes,
campestanes, poriferastanes, stigmastanes, gorgostanes, lanostanes,
cycloartanes, as well as sterol or zoosterol derivatives of any of
the foregoing, and their biological intermediates and precursors,
which may include, for example, cholesterol, lanosterol,
stigmastanol, dihydrolanosterol, zymosterol, zymostenol,
desmosterol, 7-dehydrocholesterol, and mixtures and derivatives
thereof.
[0184] Examples of non-amino acid non-cationic lipids include
pegylated cholesterols, and cholestane 3-oxo(C1-22acyl) derivatives
such as cholesteryl acetate, cholesteryl arachidonate, cholesteryl
butyrate, cholesteryl hexanoate, cholesteryl caprylate, cholesteryl
n-decanoate, cholesteryl dodecanoate, cholesteryl myristate,
cholesteryl palmitate, cholesteryl behenate, cholesteryl stearate,
cholesteryl nervonate, cholesteryl pelargonate, cholesteryl
n-valerate, cholesteryl oleate, cholesteryl elaidate, cholesteryl
erucate, cholesteryl heptanoate, cholesteryl linolelaidate,
cholesteryl linoleate, and mixtures and derivatives thereof.
[0185] Examples of non-amino acid non-cationic lipids include
compounds derived from plant sterols including phytosterols,
beta-sitosterol, campesterol, ergosterol, brassicasterol,
delta-7-stigmasterol, delta-7-avenasterol, and mixtures and
derivatives thereof.
[0186] Examples of non-amino acid non-cationic lipids include bile
acids, cholic acid, chenodeoxycholic acid, glycocholic acid,
taurocholic acid, deoxycholic acid, lithocholic acid,
methyl-lithocholic acid, and mixtures and derivatives thereof.
[0187] Examples of non-amino acid non-cationic lipids include
compounds derived from steroids including glucocorticoids,
cortisol, hydrocortisone, 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, and mixtures and derivatives thereof.
[0188] Examples of non-amino acid non-cationic lipids include
compounds derived from steroids including androgens, testosterone,
dihydrotestosterone, androstenediol, androstenedione,
androstenedione, 3.alpha.,5.alpha.-androstanediol, and mixtures and
derivatives thereof.
[0189] Examples of non-amino acid non-cationic lipids include
compounds derived from steroids including estrogens, estriols,
estrones, estradiols, and mixtures and derivatives thereof.
[0190] Examples of non-amino acid non-cationic lipids include
compounds derived from lumisterol and vitamin D compounds.
[0191] Examples of non-amino acid non-cationic lipids include
lipids having tails ranging from C10:0 to C22:6, for example, DDPE
(C10:0) (CAS 253685-27-7), DLPE (C12:0) (CAS 59752-57-7), DSPE
(C18:0) (CAS 1069-79-0), DOPE (C18:1) (CAS 4004-05-1), DLinPE
(C18:2) (CAS 20707-71-5), DLenPE (C18:3) (CAS 34813-40-6), DARAPE
(C20:4) (CAS 5634-86-6), DDHAPE (C22:6) (CAS 123284-81-1), DPhPE
(16:0[(CH.sub.3).sub.4]) (CAS 201036-16-0).
[0192] Examples of non-amino acid anionic lipids include
phosphatidylserine, phosphatidic acid, phosphatidylcholine,
platelet-activation factor (PAF), phosphatidylethanolamine,
phosphatidyl-DL-glycerol, phosphatidylinositol,
phosphatidylinositol (pi(4)p, pi(4,5)p2), cardiolipin (sodium
salt), lysophosphatides, hydrogenated phospholipids,
sphingoplipids, gangliosides, phytosphingosine, sphinganines,
pharmaceutically acceptable salts thereof, and mixtures
thereof.
Compositions and Formulations for Administration
[0193] The nucleic acid compositions and formulations of this
invention may be administered by various routes, for example, to
effect systemic delivery via intravenous, parenteral, or
intraperitoneal routes. In some embodiments, an siRNA may be
delivered intracellularly, for example, in cells of a target tissue
such as lung or liver, or in inflamed tissues. Included within this
disclosure are compositions and methods for delivery of an siRNA
agent by removing cells of a subject, delivering an siRNA agent to
the removed cells, and reintroducing the cells into a subject. In
some embodiments, this invention provides a method for delivery of
siRNA in vivo. A nucleic acid composition may be administered
intravenously, subcutaneously, or intraperitoneally to a subject.
In some embodiments, the invention provides methods for in vivo
delivery of interfering RNA to the lung of a mammalian subject.
[0194] In some embodiments, this invention provides a method of
treating a disease or disorder in a mammalian subject. A
therapeutically effective amount of a composition of this invention
containing an interfering RNA, a lipopeptide, and optionally a
non-cationic lipid, a polymeric lipid, and one or more
delivery-enhancing components or excipients may be administered to
a subject having a disease or disorder associated with expression
or overexpression of a gene that can be reduced, decreased,
downregulated, or silenced by the composition.
[0195] This invention encompasses methods for treating a disease of
the lung such as respiratory distress, asthma, cystic fibrosis,
pulmonary fibrosis, chronic obstructive pulmonary disease,
bronchitis, or emphysema, by administering to the subject a
therapeutically effective amount of a composition.
[0196] This invention encompasses methods for treating rheumatoid
arthritis, liver disease, encephalitis, bone fracture, heart
disease, viral disease including hepatitis and influenza, or
cancer.
[0197] Compositions and formulations of this disclosure may be used
for delivery of drug agents or biologically active agents to a
variety of cells in vitro. Examples of cells for which in vitro
delivery is encompassed include epithelial cells such as A549,
immortal cell lines such as HeLa, hepatoma cells such as HepG2, rat
gliosarcoma cells such as 9L/LacZ, human monocyte cells such as
THP-1, Madin-Darby canine kidney cells (MDCK), various fibroblast
cell lines, and primary cells in culture in the presence or absence
of various sera, among others.
[0198] Compositions and formulations of this disclosure may be used
for delivery of drug agents or biologically active agents to a
variety of cells, tissues or organs in vivo. Modalities for
delivering an agent in vivo include topical, enteral, and
parenteral routes. Examples of modalities for delivering an agent
in vivo include inhalation of particles or droplets, delivery of
nasal or nasal-pharngyl drops, particles, or suspensions,
transdermal and transmucosal routes, as well as injection or
infusion by intramuscular, subcutaneous, intravenous,
intraarterial, intracardiac, intrathecal, intraosseus,
intraperitoneal, and epidural routes.
[0199] In some embodiments, an agent can be administered ex vivo by
direct exposure to cells, tissues or organs originating from a
mammalian subject.
[0200] A drug agent or biologically active agent to be delivered
using a composition or formulation of this disclosure may be found
in any form including, for example, a pure form, a crystalline
form, a solid form, a nanoparticle, a condensed form, a complexed
form, or a conjugated form.
[0201] This invention also provides methods for the delivery of one
or more RNAi-inducing entities to organs and tissues within the
body of a mammal. In some embodiments, compositions containing an
RNAi-inducing entity, one or more amino acid lipids, and one or
more additional lipid components are introduced by various routes
to be transported within the body and taken up by cells in one or
more organs or tissues, where expression of a target transcript is
modulated.
[0202] Ribonucleic acid agents useful for this invention may be
targeted to various genes. Examples of human genes suitable as
targets include TNF, PLK1, BIRC5, APOB, FLT1, the VEGF family, the
ERBB family, the PDGFR family, BCR-ABL, and the MAPK family, among
others. Examples of human genes suitable as targets and nucleic
acid sequences thereto include those disclosed in PCT/US08/55333,
PCT/US08/55339, PCT/US08/55340, PCT/US08/55341, PCT/US08/55350,
PCT/US08/55353, PCT/US08/55356, PCT/US08/55357, PCT/US08/55360,
PCT/US08/55362, PCT/US08/55365, PCT/US08/55366, PCT/US08/55369,
PCT/US08/55370, PCT/US08/55371, PCT/US08/55372, PCT/US08/55373,
PCT/US08/55374, PCT/US08/55375, PCT/US08/55376, PCT/US08/55377,
PCT/US08/55378, PCT/US08/55380, PCT/US08/55381, PCT/US08/55382,
PCT/US08/55383, PCT/US08/55385, PCT/US08/55386, PCT/US08/55505,
PCT/US08/55511, PCT/US08/55515, PCT/US08/55516, PCT/US08/55519,
PCT/US08/55524, PCT/US08/55526, PCT/US08/55527, PCT/US08/55532,
PCT/US08/55533, PCT/US08/55542, PCT/US08/55548, PCT/US08/55550,
PCT/US08/55551, PCT/US08/55554, PCT/US08/55556, PCT/US08/55560,
PCT/US08/55563, PCT/US08/55597, PCT/US08/55599, PCT/US08/55601,
PCT/US08/55603, PCT/US08/55604, PCT/US08/55606, PCT/US08/55608,
PCT/US08/55611, PCT/US08/55612, PCT/US08/55615, PCT/US08/55618,
PCT/US08/55622, PCT/US08/55625, PCT/US08/55627, PCT/US08/55631,
PCT/US08/55635, PCT/US08/55644, PCT/US08/55649, PCT/US08/55651,
PCT/US08/55662, PCT/US08/55672, PCT/US08/55676, PCT/US08/55678,
PCT/US08/55695, PCT/US08/55697, PCT/US08/55698, PCT/US08/55701,
PCT/US08/55704, PCT/US08/55708, PCT/US08/55709, PCT/US08/55711,
U.S. Patent Application No. 61/086,435, and U.S. Patent Application
No. 61/086,445.
[0203] The compositions and methods of the invention may be
administered to subjects by a variety of mucosal administration
modes, including by oral, rectal, vaginal, intranasal,
intrapulmonary, or transdermal delivery, or by topical delivery to
the eyes, ears, skin or other mucosal surfaces. In some aspects of
this invention, the mucosal tissue layer includes an epithelial
cell layer. The epithelial cell can be pulmonary, tracheal,
bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal.
Compositions of this invention can be administered using
conventional actuators such as mechanical spray devices, as well as
pressurized, electrically activated, or other types of
actuators.
[0204] Compositions of this invention may be administered in an
aqueous solution as a nasal or pulmonary spray and may be dispensed
in spray form by a variety of methods known to those skilled in the
art. Pulmonary delivery of a composition of this invention may be
achieved by administering the composition in the form of drops,
particles, or spray, which can be, for example, aerosolized,
atomized, or nebulized. Pulmonary delivery may be performed by
administering the composition in the form of drops, particles, or
spray, via the nasal or bronchial passages. Particles of the
composition, spray, or aerosol can be in a either liquid or solid
form. Preferred systems for dispensing liquids as a nasal spray are
disclosed in U.S. Pat. No. 4,511,069. Such formulations may be
conveniently prepared by dissolving compositions according to the
present invention in water to produce an aqueous solution, and
rendering said solution sterile. The formulations may be presented
in multi-dose containers, for example in the sealed dispensing
system disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal
spray delivery systems have been described in Transdermal Systemic
Medication, Y. W. Chien ed., Elsevier Publishers, New York, 1985;
and in U.S. Pat. No. 4,778,810. Additional aerosol delivery forms
may include, for example, compressed air-, jet-, ultrasonic-, and
piezoelectric nebulizers, which deliver the biologically active
agent dissolved or suspended in a pharmaceutical solvent, for
example, water, ethanol, or mixtures thereof.
[0205] Nasal and pulmonary spray solutions of the present invention
typically comprise the drug or drug to be delivered, optionally
formulated with a surface active agent, such as a nonionic
surfactant (e.g., polysorbate-80), and one or more buffers. In some
embodiments of the present invention, the nasal spray solution
further comprises a propellant. The pH of the nasal spray solution
may be from about pH 6.8 to 7.2. The pharmaceutical solvents
employed can also be a slightly acidic aqueous buffer of pH 4-6.
Other components may be added to enhance or maintain chemical
stability, including preservatives, surfactants, dispersants, or
gases.
[0206] In some embodiments, this invention is a pharmaceutical
product which includes a solution containing a composition of this
invention and an actuator for a pulmonary, mucosal, or intranasal
spray or aerosol.
[0207] A dosage form of the composition of this invention can be
liquid, in the form of droplets or an emulsion, or in the form of
an aerosol.
[0208] A dosage form of the composition of this invention can be
solid, which can be reconstituted in a liquid prior to
administration. The solid can be administered as a powder. The
solid can be in the form of a capsule, tablet or gel.
[0209] To formulate compositions for pulmonary delivery within the
present invention, the biologically active agent can be combined
with various pharmaceutically acceptable additives or
delivery-enhancing components, as well as a base or carrier for
dispersion of the active agent(s). Examples of additives or
delivery-enhancing components include pH control agents such as
arginine, sodium hydroxide, glycine, hydrochloric acid, citric
acid, and mixtures thereof. Other additives or delivery-enhancing
components include local anesthetics (e.g., benzyl alcohol),
isotonizing agents (e.g., sodium chloride, mannitol, sorbitol),
adsorption inhibitors (e.g., Tween 80), solubility enhancing agents
(e.g., cyclodextrins and derivatives thereof), stabilizers (e.g.,
serum albumin), and reducing agents (e.g., glutathione). When the
composition for mucosal delivery is a liquid, the tonicity of the
formulation, as measured with reference to the tonicity of 0.9%
(w/v) physiological saline solution taken as unity, is typically
adjusted to a value at which no substantial, irreversible tissue
damage will be induced in the mucosa at the site of administration.
Generally, the tonicity of the solution is adjusted to a value of
about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to
1.7.
[0210] The biologically active agent may be dispersed in a base or
vehicle, which may comprise a hydrophilic compound having a
capacity to disperse the active agent and any desired additives.
The base may be selected from a wide range of suitable carriers,
including but not limited to, copolymers of polycarboxylic acids or
salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with
other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.),
hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives such as
hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural
polymers such as chitosan, collagen, sodium alginate, gelatin,
hyaluronic acid, and nontoxic metal salts thereof. A biodegradable
polymer may be selected as a base or carrier, for example,
polylactic acid, poly(lactic acid-glycolic acid) copolymer,
polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)
copolymer and mixtures thereof. Alternatively or additionally,
synthetic fatty acid esters such as polyglycerin fatty acid esters,
sucrose fatty acid esters, etc., can be employed as carriers.
Hydrophilic polymers and other carriers can be used alone or in
combination, and enhanced structural integrity can be imparted to
the carrier by partial crystallization, ionic bonding, crosslinking
and the like. The carrier can be provided in a variety of forms,
including, fluid or viscous solutions, gels, pastes, powders,
microspheres and films for direct application to the nasal mucosa.
The use of a selected carrier in this context may result in
promotion of absorption of the biologically active agent.
[0211] The biologically active agent can be combined with the base
or carrier according to a variety of methods, and release of the
active agent may be by diffusion, disintegration of the carrier, or
associated formulation of water channels. In some circumstances,
the active agent is dispersed in microcapsules (microspheres) or
nanocapsules (nanospheres) prepared from a suitable polymer, for
example, isobutyl 2-cyanoacrylate (see, e.g., Michael, et al., J.
Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatible
dispersing medium applied to the nasal mucosa, which yields
sustained delivery and biological activity over a protracted
time.
[0212] Formulations for mucosal, nasal, or pulmonary delivery may
contain a hydrophilic low molecular weight compound as a base or
excipient. Such hydrophilic low molecular weight compounds provide
a passage medium through which a water-soluble active agent, such
as a physiologically active peptide or protein, may diffuse through
the base to the body surface where the active agent is absorbed.
The hydrophilic low molecular weight compound optionally absorbs
moisture from the mucosa or the administration atmosphere and
dissolves the water-soluble active peptide. The molecular weight of
the hydrophilic low molecular weight compound is generally not more
than 10,000 and preferably not more than 3,000. Examples of
hydrophilic low molecular weight compounds include polyol
compounds, such as oligo-, di- and monosaccarides including
sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose,
D-xylose, D-mannose, D-galactose, lactulose, cellobiose,
gentibiose, glycerin, polyethylene glycol, and mixtures thereof.
Further examples of hydrophilic low molecular weight compounds
include N-methylpyrrolidone, alcohols (e.g., oligovinyl alcohol,
ethanol, ethylene glycol, propylene glycol, etc.), and mixtures
thereof.
[0213] The compositions of this invention may alternatively contain
as pharmaceutically acceptable carriers substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, and wetting agents,
for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, and mixtures thereof. For solid
compositions, conventional nontoxic pharmaceutically acceptable
carriers can be used which include, for example, pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharin, talcum, cellulose, glucose, sucrose, magnesium
carbonate, and the like.
[0214] In certain embodiments of the invention, the biologically
active agent may be administered in a time release formulation, for
example in a composition which includes a slow release polymer. The
active agent can be prepared with carriers that will protect
against rapid release, for example a controlled release vehicle
such as a polymer, microencapsulated delivery system or bioadhesive
gel. Prolonged delivery of the active agent, in various
compositions of the invention can be brought about by including in
the composition agents that delay absorption, for example, aluminum
monosterate hydrogels and gelatin.
[0215] Within certain embodiments of this invention, the siNA
composition may contain one or more natural or synthetic
surfactants. Certain natural surfactants are found in human lung
(pulmonary surfactant), and are a complex mixture of phospholipids
and proteins that form a monolayer at the alveolar air-liquid
interface and reduces surface tension to near zero at expiration
and prevents alveolar collapse. Over 90% (by weight) of pulmonary
surfactant is composed of phospholipids with approximately 40-80%
being DPPC and the remainder being unsaturated phosphatidylcholines
POPG, POPC and phosphatidylglycerols. The remaining 10% (by weight)
of surfactant is composed of plasma proteins and apoproteins, such
as surface proteins (SP)-A, SP-B, SP-C and SP-D.
[0216] Examples of natural surfactants that may be used in this
invention include SURVANTA.TM. (beractant), CUROSURF.TM. (poractant
alfa) and INFASURF.TM. (calfactant), and mixtures thereof.
[0217] Examples of synthetic surfactants include sinapultide; a
combination of dipalmitoylphosphatidylcholine, palmitoyloleoyl
phosphatidylglycerol and palmitic acid; SURFAXIN.TM. (lucinactant);
and EXOSURF.TM. (colfosceril); components which may contain
tyloxapol, DPPC, and hexadecanol; and mixtures thereof.
[0218] Compositions of this invention can be prepared by methods
known in the art. Methods of making the lipid compositions include
ethanol injection methods and extrusion methods using a Northern
Lipids Lipex Extruder system with stacked polycarbonate membrane
filters of defined pore size. Sonication using probe tip and bath
sonicators can be employed to produce lipid particles of uniform
size. Homogenous and monodisperse particle sizes can be obtained
without the addition of the nucleic acid component. For in vitro
transfection compositions, the nucleic acid component can be added
after the transfection agent is made and stabilized by additional
buffer components. For in vivo delivery compositions, the nucleic
acid component is part of the formulation.
[0219] A mixing procedure involving the graded substitution of
ethanol for buffer can be used to create a narrow homodisperse
particle size distribution. The lipid components and lipopeptides
can be dissolved in USP absolute ethanol and the RNA component can
be dissolved in an aqueous buffer, for example, at 0.9 mg/mL. Both
mixtures can be injected through an HPLC mixing tee into a 20 mM
citrate buffer at pH 7.2. The initial concentration of ethanol may
be 90% for the lipids and 0% for the RNA component. After mixing
into a 45% ethanol mixture, the RNA-lipid particles can be
immediately diluted into citrate buffer with a final concentration
of ethanol at 30%. The ethanol and citrate buffer can be exchanged
for PBS, pH 7.2 by overnight dialysis in a Pierce dialysis cassette
with a 2K MWCO membrane. Particle sizing and PAGE gel analysis of
each siRNA lipid particle can be performed to confirm successful
entrapment. PAGE gel results can confirm that an siRNA is intact
after exposure to multiple processing steps.
[0220] The nucleic acid component, lipopeptides, and any additional
components may be mixed together first in a suitable medium such as
a cell culture medium, after which one or more additional lipids or
compounds may be added to the mixture. Alternatively, the
lipopeptides can be mixed together first in a suitable medium such
as a cell culture medium, after which the nucleic acid component
can be added.
[0221] Within certain embodiments of the invention, a dsRNA is
admixed with one or more lipopeptides, or a combination of one or
more lipopeptides and non-cationic lipids.
[0222] The interfering RNA agent may also be complexed with, or
conjugated to a lipopeptide or a polymeric lipid, and admixed with
one or more non-cationic lipids, or a combination of one or more
non-cationic and cationic lipids.
[0223] An interfering RNA agent and a lipopeptide may be mixed
together first, followed by the addition of one or more
non-cationic lipids, or a combination of non-cationic and cationic
lipids added in a suitable medium such as a cell culture medium.
Alternatively, the lipopeptides and lipid components may be mixed
first, followed by the addition of the RNA agent in a suitable
medium.
RNA Therapeutics and RNA Interference
[0224] This invention provides compositions and methods for
modulating gene expression by RNA interference. A composition of
this invention can deliver a ribonucleic acid agent to a cell which
can produce the response of RNAi. Examples of nucleic acid agents
useful for this invention include double-stranded nucleic acids,
modified or degradation-resistant nucleic acids, RNA, siRNA, siNA,
mdRNA, shRNA, single-stranded nucleic acids, DNA-RNA chimeras,
antisense nucleic acids, and ribozymes. As used herein, the terms
siRNA, siNA, and shRNA include precursors of siRNA, siNA, and
shRNA, respectively. For example, the term siRNA includes an RNA or
double-stranded RNA that is suitable as a dicer substrate.
[0225] Meroduplex RNA (mdRNA) is described in U.S. Provisional
Application No. 60/934,930, as well as International Publication
No. WO/2007/056153.
[0226] Ribonucleic acid agents useful for this invention may be
targeted to various genes. For example, a siRNA agent of this
invention may have a sequence that is complementary to a region of
a TNF-alpha gene. In some embodiments of this invention, compounds
and compositions are useful to regulate expression of tumor
necrosis factor-.alpha.(TNF-.alpha.). TNF-.alpha. can be linked,
for example, to inflammatory processes which occur in pulmonary
diseases, and can have anti-inflammatory effects. Blocking
TNF-.alpha. by delivery of a composition of this invention can be
useful to treat or prevent the signs and/or symptoms of rheumatoid
arthritis. This invention provides compositions and methods for
modulating expression and activity of TNF-.alpha. by RNA
interference.
[0227] Expression and/or activity of TNF-.alpha. can be modulated
by delivering to a cell, for example, the siRNA molecule Inm-4.
Inm-4 is a double stranded 21-nt siRNA molecule with sequence
homology to the mouse TNF-.alpha. gene Inm-4 has a 3' dTdT overhang
on the sense strand and a 3' dAdT overhang on the antisense strand.
The primary structure of Inm-4 is:
TABLE-US-00005 sense 5'-CCGUCAGCCGAUUUGCUAUdTdT (SEQ ID NO: 301)
antisense 5'-AUAGCAAAUCGGCUGACGGdTdT (SEQ ID NO: 302)
[0228] Expression and/or activity of TNF-.alpha. can be modulated
by delivering to a cell, for example, the siRNA molecule LC20. LC20
is a double stranded 21-nt siRNA molecule with sequence homology to
the human TNF-.alpha. gene. LC20 is directed against the 3'-UTR
region of human TNF-.alpha.. LC 20 has 19 base pairs with a 3' dTdT
overhang on the sense strand and a 3' dAdT overhang on the
antisense strand. The molecular weight of the sodium salt form is
14,298. The primary structure of LC20 is:
TABLE-US-00006 sense (5') GGGUCGGAACCCAAGCUUAdTdT (SEQ ID NO: 303)
antisense (5') UAAGCUUGGGUUCCGACCCdTdA (SEQ ID NO: 304)
[0229] A .beta.-galactoside reporter cell line was used to assay
the RNAi activity of various formulations. The structure of Lac-Z
is:
TABLE-US-00007 Sense: CN2938. 5'-CUACACAAAUCAGCGAUUUdTdT-3' (SEQ ID
NO: 305) Antisense: CN2939. (SEQ ID NO: 306)
5'-AAAUCGCUGAUUUGUGUAGdTdC-3'
[0230] A siRNA of this invention may have a sequence that is
complementary to a region of a viral gene. For example, some
compositions and methods of this invention are useful to regulate
expression of the viral genome of an influenza.
[0231] In this context, this invention provides compositions and
methods for modulating expression and infectious activity of an
influenza by RNA interference. Expression and/or activity of an
influenza can be modulated by delivering to a cell, for example, a
short interfering RNA molecule having a sequence that is
complementary to a region of a RNA polymerase subunit of an
influenza. For example, in Table 5 are shown double-stranded siRNA
molecules with sequence homology to an RNA polymerase subunit of an
influenza.
TABLE-US-00008 TABLE 5 Double-Stranded siRNA Molecules Targeted to
Influenza siRNA Subunit SEQUENCE G3789 PB2 (SEQ ID NO 307)
CGGGACUCUAGCAUACUUAdTdT (SEQ ID NO 308) UAAGUAUGCUAGAGUCCCGdTdT
G3807 PB2 (SEQ ID NO 309) ACUGACAGCCAGACAGCGAdTdT (SEQ ID NO 310)
UCGCUGUCUGGCUGUCAGUdTdT G3817 PB2 (SEQ ID NO 311)
AGACAGCGACCAAAAGAAUdTdT (SEQ ID NO 312) AUUCUUUUGGUCGCUGUCUdTdT
G6124 PB1 (SEQ ID NO 313) AUGAAGAUCUGUUCCACCAdTdT (SEQ ID NO 314)
UGGUGGAACAGAUCUUCAUdTdT G6129 PB1 (SEQ ID NO 315)
GAUCUGUUCCACCAUUGAAdTdT (SEQ ID NO 316) UUCAAUGGUGGAACAGAUCdTdT
G8282 PA (SEQ ID NO 317) GCAAUUGAGGAGUGCCUGAdTdT (SEQ ID NO 318)
UCAGGCACUCCUCAAUUGCdTdT G8286 PA (SEQ ID NO 319)
UUGAGGAGUGCCUGAUUAAdTdT (SEQ ID NO 320) UUAAUCAGGCACUCCUCAAdTdT
G1498 NP (SEQ ID NO 321) GGAUCUUAUUUCUUCGGAGdTdT (SEQ ID NO 322)
CUCCGAAGAAAUAAGAUCCdTdT
[0232] A siRNA of this invention may have a sequence that is
complementary to a region of a RNA polymerase subunit of an
influenza.
[0233] This invention provides compositions and methods to
administer siNAs directed against a mRNA of an influenza, which
effectively down-regulates an influenza RNA and thereby reduces,
prevents, or ameliorates an influenza infection.
[0234] In some embodiments, this invention provides compositions
and methods for inhibiting expression of a target transcript in a
subject by administering to the subject a composition containing an
effective amount of an RNAi-inducing compound such as a short
interfering oligonucleotide molecule, or a precursor thereof. RNAi
uses small interfering RNAs (siRNAs) to target messenger RNA
(mRNAs) and attenuate translation. A siRNA as used in this
invention may be a precursor for dicer processing such as, for
example, a long dsRNA processed into a siRNA. This invention
provides methods of treating or preventing diseases or conditions
associated with expression of a target transcript or activity of a
peptide or protein encoded by the target transcript.
[0235] A therapeutic strategy based on RNAi can be used to treat a
wide range of diseases by shutting down the growth or function of a
virus or microorganism, as well as by shutting down the function of
an endogenous gene product in the pathway of the disease.
[0236] In some embodiments, this invention provides novel
compositions and methods for delivery of RNAi-inducing entities
such as short interfering oligonucleotide molecules, and precursors
thereof. In particular, this invention provides compositions
containing an RNAi-inducing entity which is targeted to one or more
transcripts of a cell, tissue, and/or organ of a subject.
[0237] A siRNA can be two RNA strands having a region of
complementarity about 19 nucleotides in length. A siRNA optionally
includes one or two single-stranded overhangs or loops.
[0238] A shRNA can be a single RNA strand having a region of
self-complementarity. The single RNA strand may form a hairpin
structure with a stem and loop and, optionally, one or more
unpaired portions at the 5' and/or 3' portion of the RNA.
[0239] The active therapeutic agent can be a chemically-modified
siNA with improved resistance to nuclease degradation in vivo,
and/or improved cellular uptake, which retains RNAi activity.
[0240] A siRNA agent of this invention may have a sequence that is
complementary to a region of a target gene. A siRNA of this
invention may have 29-50 base pairs, for example, a dsRNA having a
sequence that is complementary to a region of a target gene.
Alternately, the double-stranded nucleic acid can be a dsDNA.
[0241] In some embodiments, the active agent can be a short
interfering nucleic acid (siNA), short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA, or short hairpin RNA
(shRNA) that can modulate expression of a gene product.
[0242] Comparable methods and compositions are provided that target
expression of one or more different genes associated with a
particular disease condition in a subject, including any of a large
number of genes whose expression is known to be aberrantly
increased as a causal or contributing factor associated with the
selected disease condition.
[0243] The RNAi-inducing compound of this invention can be
administered in conjunction with other known treatments for a
disease condition.
[0244] In some embodiments, this invention features compositions
containing a small nucleic acid molecule, such as short interfering
nucleic acid, a short interfering RNA, a double-stranded RNA, a
micro-RNA, or a short hairpin RNA, admixed or complexed with, or
conjugated to, a delivery-enhancing compound.
[0245] As used herein, the terms "short interfering nucleic acid,"
"siNA," "short interfering RNA," "siRNA," "short interfering
nucleic acid molecule," "short interfering oligonucleotide
molecule," and "chemically-modified short interfering nucleic acid
molecule," refer to any nucleic acid molecule capable of inhibiting
or down regulating gene expression or viral replication, for
example, by mediating RNA interference (RNAi) or gene silencing in
a sequence-specific manner.
[0246] In some embodiments, the siNA is a double-stranded
polynucleotide molecule comprising self-complementary sense and
antisense regions, wherein the antisense region comprises a
nucleotide sequence that is complementary to a nucleotide sequence
in a target ribonucleic acid molecule for down regulating
expression, or a portion thereof, and the sense region comprises a
nucleotide sequence corresponding to (i.e., which is substantially
identical in sequence to) the target ribonucleic acid sequence or
portion thereof
[0247] "siNA" means a small interfering nucleic acid, for example a
siRNA, that is a short-length double-stranded nucleic acid, or
optionally a longer precursor thereof. The length of useful siNAs
within this invention will in some embodiments be preferred at a
length of approximately 20 to 50 bp. However, there is no
particular limitation to the length of useful siNAs, including
siRNAs. For example, siNAs can initially be presented to cells in a
precursor form that is substantially different than a final or
processed form of the siNA that will exist and exert gene silencing
activity upon delivery, or after delivery, to the target cell.
Precursor forms of siNAs may, for example, include precursor
sequence elements that are processed, degraded, altered, or cleaved
at or after the time of delivery to yield a siNA that is active
within the cell to mediate gene silencing. In some embodiments,
useful siNAs will have a precursor length, for example, of
approximately 100-200 base pairs, or 50-100 base pairs, or less
than about 50 base pairs, which will yield an active, processed
siNA within the target cell. In other embodiments, a useful siNA or
siNA precursor will be approximately 10 to 49 bp, or 15 to 35 bp,
or about 21 to 30 by in length.
[0248] In some embodiments of this invention, polynucleotide
delivery-enhancing polypeptides are used to facilitate delivery of
larger nucleic acid molecules than conventional siNAs, including
large nucleic acid precursors of siNAs. For example, the methods
and compositions herein may be employed for enhancing delivery of
larger nucleic acids that represent "precursors" to desired siNAs,
wherein the precursor amino acids may be cleaved or otherwise
processed before, during or after delivery to a target cell to form
an active siNA for modulating gene expression within the target
cell.
[0249] For example, a siNA precursor polynucleotide may be selected
as a circular, single-stranded polynucleotide, having two or more
loop structures and a stem comprising self-complementary sense and
antisense regions, wherein the antisense region comprises a
nucleotide sequence that is complementary to a nucleotide sequence
in a target nucleic acid molecule or a portion thereof, and the
sense region having nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof, and wherein the
circular polynucleotide can be processed either in vivo or in vitro
to generate an active siNA molecule capable of mediating RNAi.
[0250] siNA molecules of this invention, particularly non-precursor
forms, can be less than 30 base pairs, or about 17-19 bp, or 19-21
bp, or 21-23 bp.
[0251] siRNAs can mediate selective gene silencing in the mammalian
system. Hairpin RNAs, with a short loop and 19 to 27 base pairs in
the stem, also selectively silence 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.
[0252] 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 within the region
complementary to the antisense strand of the siRNA duplex. siRNA
duplexes of 21 nucleotides are typically most active when
containing two-nucleotide 3'-overhangs.
[0253] Replacing the 3'-overhanging segments of a 21-mer siRNA
duplex having 2-nucleotide 3' overhangs with deoxyribonucleotides
may not have an adverse effect on RNAi activity. Replacing up to 4
nucleotides on each end of the siRNA with deoxyribonucleotides can
be tolerated whereas complete substitution with
deoxyribonucleotides may result in no RNAi activity.
[0254] Alternatively, the siNAs can be delivered as single or
multiple transcription products expressed by a polynucleotide
vector encoding the single or multiple siNAs and directing their
expression within target cells. In these embodiments the
double-stranded portion of a final transcription product of the
siRNAs to be expressed within the target cell can be, for example,
15 to 49 bp, 15 to 35 bp, or about 21 to 30 by long.
[0255] In some embodiments of this invention, the double-stranded
region of siNAs in which two strands are paired may contain bulge
or mismatched portions, or both. Double-stranded portions of siNAs
in which two strands are paired are not limited to completely
paired nucleotide segments, and may contain nonpairing portions due
to, for example, mismatch (the corresponding nucleotides not being
complementary), bulge (lacking in the corresponding complementary
nucleotide on one strand), or overhang. Nonpairing portions can be
contained to the extent that they do not interfere with siNA
formation. In some embodiments, a "bulge" may be 1 to 2 nonpairing
nucleotides, and the double-stranded region of siNAs in which two
strands pair up may contain from about 1 to 7, or about 1 to 5
bulges. In addition, "mismatch" portions contained in the
double-stranded region of siNAs may be present in numbers from
about 1 to 7, or about 1 to 5. Most often in the case of
mismatches, one of the nucleotides is guanine, and the other is
uracil. Such mismatching may be attributable, for example, to a
mutation from C to T, G to A, or mixtures thereof, in a
corresponding DNA coding for sense RNA, but other causes are also
contemplated.
[0256] The terminal structure of siNAs of this invention may be
either blunt or cohesive (overhanging) as long as the siNA retains
its activity to silence expression of target genes. The cohesive
(overhanging) end structure is not limited to the 3' overhang, but
includes the 5' overhanging structure as long as it retains
activity for inducing gene silencing. In addition, the number of
overhanging nucleotides is not limited to 2 or 3 nucleotides, but
can be any number of nucleotides as long as it retains activity for
inducing gene silencing. For example, overhangs may comprise from 1
to about 8 nucleotides, or from 2 to 4 nucleotides.
[0257] The length of siNAs having cohesive (overhanging) end
structure may be expressed in terms of the paired duplex portion
and any overhanging portion at each end. For example, a 25/27-mer
siNA duplex with a 2-bp 3' antisense overhang has a 25-mer sense
strand and a 27-mer antisense strand, where the paired portion has
a length of 25 bp.
[0258] Any overhang sequence may have low specificity to a target
gene, and may not be complementary (antisense) or identical (sense)
to the target gene sequence. As long as the siNA retains activity
for gene silencing, it may contain in the overhang portion a low
molecular weight structure, for example, a natural RNA molecule
such as a tRNA, an rRNA, a viral RNA, or an artificial RNA
molecule.
[0259] The terminal structure of the siNAs may have a stem-loop
structure in which ends of one side of the double-stranded nucleic
acid are connected by a linker nucleic acid, for example, a linker
RNA. The length of the double-stranded region (stem portion) can
be, for example, 15 to 49 bp, or 15 to 35 bp, or about 21 to 30 by
long. Alternatively, the length of the double-stranded region that
is a final transcription product of siNAs to be expressed in a
target cell may be, for example, approximately 15 to 49 bp, or 15
to 35 bp, or about 21 to 30 by long.
[0260] The siNA can contain a single stranded polynucleotide having
a nucleotide sequence complementary to a nucleotide sequence in a
target nucleic acid molecule, or a portion thereof, wherein the
single stranded polynucleotide can contain a terminal phosphate
group, such as a 5'-phosphate (see e.g. Martinez, et al., Cell.
110:563-574, 2002, and Schwarz, et al., Molecular Cell 10:537-568,
2002, or 5',3'-diphosphate.
[0261] As used herein, the term siNA molecule is not limited to
molecules containing only naturally-occurring RNA or DNA, but also
encompasses chemically-modified nucleotides and non-nucleotides. In
some embodiments, the short interfering nucleic acid molecules of
the invention lack 2'-hydroxy (2'-OH) containing nucleotides. In
some embodiments, short interfering nucleic acids do not require
the presence of nucleotides having a 2'-hydroxy group for mediating
RNAi and as such, short interfering nucleic acid molecules of this
invention optionally do not include any ribonucleotides (e.g.,
nucleotides having a 2'-OH group). siNA molecules that do not
require the presence of ribonucleotides within the siNA molecule to
support RNAi can, however, have an attached linker or linkers or
other attached or associated groups, moieties, or chains containing
one or more nucleotides with 2'-OH groups. siNA molecules can
comprise ribonucleotides in at least about 5, 10, 20, 30, 40, or
50% of the nucleotide positions.
[0262] As used herein, the term siNA encompasses nucleic acid
molecules that are capable of mediating sequence specific RNAi such
as, for example, short interfering RNA (siRNA) molecules,
double-stranded RNA (dsRNA) molecules, micro-RNA molecules, short
hairpin RNA (shRNA) molecules, short interfering oligonucleotide
molecules, short interfering nucleic acid molecules, short
interfering modified oligonucleotide molecules, chemically-modified
siRNA molecules, and post-transcriptional gene silencing RNA
(ptgsRNA) molecules, among others.
[0263] In some embodiments, siNA molecules comprise separate sense
and antisense sequences or regions, wherein the sense and antisense
regions are covalently linked by nucleotide or non-nucleotide
linker molecules, or are non-covalently linked by ionic
interactions, hydrogen bonding, van der waals interactions,
hydrophobic interactions, and/or stacking interactions.
[0264] "Antisense RNA" is an RNA strand having a sequence
complementary to a target gene mRNA, that can induce RNAi by
binding to the target gene mRNA.
[0265] "Sense RNA" is an RNA strand having a sequence complementary
to an antisense RNA, and anneals to its complementary antisense RNA
to form a siRNA.
[0266] As used herein, the term "RNAi construct" or "RNAi
precursor" refers to an RNAi-inducing compound such as small
interfering RNAs (siRNAs), hairpin RNAs, and other RNA species
which can be cleaved in vivo to form a siRNA. RNAi precursors
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.
[0267] A 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 which
binds to a 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.
[0268] siNAs for use within the invention can be assembled from two
separate oligonucleotides, where one strand is the sense strand and
the other is the antisense strand, wherein the antisense and sense
strands are self-complementary (i.e., each strand comprises
nucleotide sequence that is complementary to nucleotide sequence in
the other strand; such as where the antisense strand and sense
strand form a duplex or double stranded structure, for example
wherein the double stranded region is about 19 base pairs). The
antisense strand may comprise a nucleotide sequence that is
complementary to a nucleotide sequence in a target nucleic acid
molecule or a portion thereof, and the sense strand may comprise a
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. Alternatively, the siNA can be
assembled from a single oligonucleotide, where the
self-complementary sense and antisense regions of the siNA are
linked by means of a nucleic acid-based or non-nucleic acid-based
linker(s).
[0269] In some embodiments, siNAs for intracellular delivery can be
a polynucleotide with a duplex, asymmetric duplex, hairpin or
asymmetric hairpin secondary structure, having self-complementary
sense and antisense regions, wherein the antisense region comprises
a nucleotide sequence that is complementary to a nucleotide
sequence in a separate target nucleic acid molecule or a portion
thereof, and the sense region comprises a nucleotide sequence
corresponding to the target nucleic acid sequence or a portion
thereof.
[0270] Examples of chemical modifications that can be made in an
siNA include phosphorothioate internucleotide linkages,
2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides,
2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides,
"acyclic" nucleotides, 5-C-methyl nucleotides, and terminal
glyceryl and/or inverted deoxy abasic residue incorporation.
[0271] The antisense region of a siNA molecule can include a
phosphorothioate internucleotide linkage at the 3'-end of said
antisense region. The antisense region can comprise about one to
about five phosphorothioate internucleotide linkages at the 5'-end
of said antisense region. The 3'-terminal nucleotide overhangs of a
siNA molecule can include ribonucleotides or deoxyribonucleotides
that are chemically-modified at a nucleic acid sugar, base, or
backbone. The 3'-terminal nucleotide overhangs can include one or
more universal base ribonucleotides. The 3'-terminal nucleotide
overhangs can comprise one or more acyclic nucleotides.
[0272] For example, a chemically-modified siNA can have 1, 2, 3, 4,
5, 6, 7, 8, or more phosphorothioate internucleotide linkages in
one strand, or can have 1 to 8 or more phosphorothioate
internucleotide linkages in each strand. The phosphorothioate
internucleotide linkages can be present in one or both
oligonucleotide strands of the siNA duplex, for example in the
sense strand, the antisense strand, or both strands.
[0273] siNA molecules can comprise one or more phosphorothioate
internucleotide linkages at the 3'-end, the 5'-end, or both of the
3'- and 5'-ends of the sense strand, the antisense strand, or in
both strands. For example, an exemplary siNA molecule can include
1, 2, 3,4, 5, or more consecutive phosphorothioate internucleotide
linkages at the 5'-end of the sense strand, the antisense strand,
or both strands.
[0274] In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or more pyrimidine phosphorothioate internucleotide
linkages in the sense strand, the antisense strand, or in both
strands.
[0275] In some embodiments, a siNA molecule includes 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or more purine phosphorothioate internucleotide
linkages in the sense strand, the antisense strand, or in both
strands.
[0276] A siNA molecule can include a circular nucleic acid
molecule, wherein the siNA is about 38 to about 70, for example,
about 38, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length,
having about 18 to about 23, for example, about 18, 19, 20, 21, 22,
or 23 base pairs, wherein the circular oligonucleotide forms a
dumbbell-shaped structure having about 19 base pairs and 2
loops.
[0277] A circular siNA molecule can contain two loop motifs,
wherein one or both loop portions of the siNA molecule is
biodegradable. For example, the loop portions of a circular siNA
molecule may be transformed in vivo to generate a double-stranded
siNA molecule with 3'-terminal overhangs, such as 3'-terminal
nucleotide overhangs comprising about 2 nucleotides.
[0278] Modified nucleotides in a siNA molecule can be in the
antisense strand, the sense strand, or both. For example, modified
nucleotides can have a Northern conformation (e.g., Northern
pseudorotation cycle; see e.g., Saenger, Principles of Nucleic Acid
Structure, Springer-Verlag ed., 1984). Examples of nucleotides
having a Northern configuration include locked nucleic acid (LNA)
nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl)
nucleotides), 2'-methoxyethoxy (MOE) nucleotides,
2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides,
2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and
2'-O-methyl nucleotides.
[0279] Chemically modified nucleotides can be resistant to nuclease
degradation while at the same time maintaining the capacity to
mediate RNAi.
[0280] The sense strand of a double stranded siNA molecule may have
a terminal cap moiety such as an inverted deoxyabasic moiety, at
the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand.
[0281] Examples of conjugates include conjugates and ligands
described in Vargeese, et al., U.S. application Ser. No.
10/427,160, filed Apr. 30, 2003, incorporated by reference herein
in its entirety, including the drawings.
[0282] In some embodiments of this invention, the conjugate may be
covalently attached to the chemically-modified siNA molecule via a
biodegradable linker. For example, the conjugate molecule may be
attached at the 3'-end of either the sense strand, the antisense
strand, or both strands of the chemically-modified siNA
molecule.
[0283] In some embodiments, the conjugate molecule is attached at
the 5'-end of either the sense strand, the antisense strand, or
both strands of the chemically-modified siNA molecule. In some
embodiments, the conjugate molecule is attached both the 3'-end and
5'-end of either the sense strand, the antisense strand, or both
strands of the chemically-modified siNA molecule, or any
combination thereof.
[0284] In some embodiments, a conjugate molecule comprises a
molecule that facilitates delivery of a chemically-modified siNA
molecule into a biological system, such as a cell.
[0285] In some embodiments, a conjugate molecule attached to the
chemically-modified siNA molecule is a polyethylene glycol, human
serum albumin, or a ligand for a cellular receptor that can mediate
cellular uptake. Examples of specific conjugate molecules
contemplated by the instant invention that can be attached to
chemically-modified siNA molecules are described in Vargeese, et
al., U.S. Patent Publication Nos. 2003/0130186 and
2004/0110296.
[0286] A siNA may be contain a nucleotide, non-nucleotide, or mixed
nucleotide/non-nucleotide linker that joins the sense region of the
siNA to the antisense region of the siNA. In some embodiments, a
nucleotide linker can be 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in
length. In some embodiments, the nucleotide linker can be a nucleic
acid aptamer. As used herein, the terms "aptamer" or "nucleic acid
aptamer" encompass a nucleic acid molecule that binds specifically
to a target molecule, wherein the nucleic acid molecule contains a
sequence that is recognized by the target molecule in its natural
setting. Alternately, an aptamer can be a nucleic acid molecule
that binds to a target molecule where the target molecule does not
naturally bind to a nucleic acid.
[0287] For example, the aptamer can be used to bind to a
ligand-binding domain of a protein, thereby preventing interaction
of the naturally occurring ligand with the protein. See, for
example, Gold, et al., Annu. Rev. Biochem. 64:763, 1995; Brody and
Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100,
2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel,
Science 287:820, 2000; and Jayasena, Clinical Chemistry 45:1628,
1999.
[0288] A non-nucleotide linker can be an abasic nucleotide,
polyether, polyamine, polyamide, peptide, carbohydrate, lipid,
polyhydrocarbon, or other polymeric compounds (e.g., polyethylene
glycols such as those having between 2 and 100 ethylene glycol
units). Specific examples include those described by Seela and
Kaiser, Nucleic Acids Res. 18:6353, 1990, and Nucleic Acids Res.
15:3113, 1987; Cload and Schepartz, J. Am. Chem. Soc. 113:6324,
1991; Richardson and Schepartz, J. Am. Chem. Soc. 113:5109, 1991;
Ma, et al., Nucleic Acids Res. 21:2585, 1993, and Biochemistry
32:1751, 1993; Durand, et al., Nucleic Acids Res. 18:6353, 1990;
McCurdy, et al., Nucleosides & Nucleotides 10:287, 1991;
Jaschke, et al., Tetrahedron Lett. 34:301-304, 1993; Ono, et al.,
Biochemistry 30:9914, 1991; Arnold, et al., International
Publication No. WO/1989/02439; Usman, et al., International
Publication No. WO/1995/06731; Dudycz, et al., International
Publication No. WO/1995/11910, and Ferentz and Verdine, J. Am.
Chem. Soc. 113:4000, 1991.
[0289] A "non-nucleotide linker" refers to a group or compound that
can be incorporated into a nucleic acid chain in the place of one
or more nucleotide units, including either sugar and/or phosphate
substitutions, and allows the remaining bases to exhibit their
enzymatic activity. The group or compound can be abasic in that it
does not contain a commonly recognized nucleotide base, such as
adenosine, guanine, cytosine, uracil or thymine, for example at the
C1 position of the sugar.
[0290] In some embodiments, modified siNA molecule can have
phosphate backbone modifications including one or more
phosphorothioate, phosphorodithioate, methylphosphonate,
phosphotriester, morpholino, amidate carbamate, carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate,
formacetal, thioformacetal, and/or alkylsilyl substitutions.
Examples of oligonucleotide backbone modifications are given in
Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and
Properties, in Modern Synthetic Methods, VCH, pp. 331-417, 1995,
and Mesmaeker, et al., Novel Backbone Replacements for
Oligonucleotides, in Carbohydrate Modifications in Antisense
Research, ACS, pp. 24-39, 1994.
[0291] siNA molecules, which can be chemically-modified, can be
synthesized by: (a) synthesis of two complementary strands of the
siNA molecule; and (b) annealing the two complementary strands
together under conditions suitable to obtain a double-stranded siNA
molecule. In some embodiments, synthesis of the complementary
portions of the siNA molecule is by solid phase oligonucleotide
synthesis, or by solid phase tandem oligonucleotide synthesis.
[0292] Oligonucleotides (e.g., certain modified oligonucleotides or
portions of oligonucleotides lacking ribonucleotides) are
synthesized using protocols known in the art, for example, as
described in Caruthers, et al., Methods in Enzymology 211:3-19,
1992; Thompson, et al., International Publication No.
WO/1999/54459; Wincott, et al., Nucleic Acids Res. 23:2677-2684,
1995; Wincott, et al., Methods Mol. Bio. 74:59, 1997; Brennan, et
al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan, U.S. Pat. No.
6,001,311. Synthesis of RNA, including certain siNA molecules of
the invention, follows general procedures as described, for
example, in Usman, et al., J. Am. Chem. Soc. 109:7845, 1987;
Scaringe, et al., Nucleic Acids Res. 18:5433, 1990; and Wincott, et
al., Nucleic Acids Res. 23:2677-2684, 1995; Wincott, et al.,
Methods Mol. Bio. 74:59, 1997.
[0293] An "asymmetric hairpin" as used herein is a linear siNA
molecule comprising an antisense region, a loop portion that can
comprise nucleotides or non-nucleotides, and a sense region that
comprises fewer nucleotides than the antisense region to the extent
that the sense region has enough complementary nucleotides to base
pair with the antisense region and form a duplex with loop.
[0294] An "asymmetric duplex" as used herein is a siNA molecule
having two separate strands comprising a sense region and an
antisense region, wherein the sense region comprises fewer
nucleotides than the antisense region to the extent that the sense
region has enough complementary nucleotides to base pair with the
antisense region and form a duplex.
[0295] To "modulate gene expression" as used herein is to
upregulate or down-regulate expression of a target gene, which can
include upregulation or downregulation of mRNA levels present in a
cell, or of mRNA translation, or of synthesis of protein or protein
subunits, encoded by the target gene.
[0296] The terms "inhibit," "down-regulate," or "reduce expression"
as used herein mean that the expression of the gene, or level of
RNA molecules or equivalent RNA molecules encoding one or more
proteins or protein subunits, or level or activity of one or more
proteins or protein subunits encoded by a target gene, is reduced
below that observed in the absence of the nucleic acid molecules
(e.g., siNA) of the invention.
[0297] "Gene silencing" as used herein refers to partial or
complete inhibition of gene expression in a cell and may also be
referred to as "gene knockdown." The extent of gene silencing may
be determined by methods known in the art, some of which are
summarized in International Publication No. WO/1999/32619.
[0298] As used herein, the terms "ribonucleic acid" and "RNA" refer
to a molecule containing at least one ribonucleotide residue. A
ribonucleotide is a nucleotide with a hydroxyl group at the 2'
position of a .beta.-D-ribo-furanose moiety. These terms include
double-stranded RNA, single-stranded RNA, isolated RNA such as
partially purified RNA, essentially pure RNA, synthetic RNA,
recombinantly produced RNA, as well as modified and altered RNA
that differs from naturally occurring RNA by the addition,
deletion, substitution, modification, and/or alteration of one or
more nucleotides. Alterations of an RNA can include addition of
non-nucleotide material, such as to the end(s) of a siNA or
internally, for example at one or more nucleotides of an RNA.
[0299] Nucleotides in an RNA molecule include non-standard
nucleotides, such as non-naturally occurring nucleotides or
chemically synthesized nucleotides or deoxynucleotides. These
altered RNAs can be referred to as analogs.
[0300] By "highly conserved sequence region" is meant, a nucleotide
sequence of one or more regions in a target gene does not vary
significantly from one generation to the other or from one
biological system to the other.
[0301] By "sense region" is meant a nucleotide sequence of a siNA
molecule having complementarity to an antisense region of the siNA
molecule. In addition, the sense region of a siNA molecule can
comprise a nucleic acid sequence having homology with a target
nucleic acid sequence.
[0302] By "antisense region" is meant a nucleotide sequence of a
siNA molecule having complementarity to a target nucleic acid
sequence. In addition, the antisense region of a siNA molecule can
include a nucleic acid sequence having complementarity to a sense
region of the siNA molecule.
[0303] By "target nucleic acid" is meant any nucleic acid sequence
whose expression or activity is to be modulated. A target nucleic
acid can be DNA or RNA.
[0304] By "complementarity" is meant that a nucleic acid can form
hydrogen bond(s) with another nucleic acid sequence either by
traditional Watson-Crick or by other non-traditional modes of
binding.
[0305] The term "biodegradable linker" as used herein, refers to a
nucleic acid or non-nucleic acid linker molecule that is designed
as a biodegradable linker to connect one molecule to another
molecule, for example, a biologically active molecule to a siNA
molecule or the sense and antisense strands of a siNA molecule. The
biodegradable linker is designed such that its stability can be
modulated for a particular purpose, such as delivery to a
particular tissue or cell type. The stability of a nucleic
acid-based biodegradable linker molecule can be variously
modulated, for example, by combinations of ribonucleotides,
deoxyribonucleotides, and chemically-modified nucleotides, such as
2'-O-methyl, 2'-fluoro, 2'-amino, 2'-.beta.-amino, 2'-C-allyl,
2'-O-allyl, and other 2'-modified or base modified nucleotides. The
biodegradable nucleic acid linker molecule can be a dimer, trimer,
tetramer or longer nucleic acid molecule, for example, an
oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can
comprise a single nucleotide with a phosphorus-based linkage, for
example, a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise
nucleic acid backbone, nucleic acid sugar, or nucleic acid base
modifications.
[0306] In connection with 2'-modified nucleotides as described
herein, by "amino" is meant 2'-NH.sub.2 or 2'-O-NH.sub.2, which can
be modified or unmodified. Such modified groups are described, for
example, in Eckstein, et al., U.S. Pat. No. 5,672,695 and
Matulic-Adamic, et al., U.S. Pat. No. 6,248,878.
[0307] Supplemental or complementary methods for delivery of
nucleic acid molecules for use within then invention are described,
for example, in Akhtar et al., Trends Cell Bio. 2:139, 1992;
"Delivery Strategies for Antisense Oligonucleotide Therapeutics,"
ed Akhtar, 1995, Maurer et al., Mol. Membr. Biol. 16:129-140, 1999;
Hofland and Huang, Handb. Exp. Pharmacol. 137:165-192, 1999; and
Lee et al., ACS Symp. Ser. 752:184-192, 2000. Sullivan, et al.,
International Publication No. WO/1994/02595, further describes
general methods for delivery of enzymatic nucleic acid
molecules.
[0308] Nucleic acid molecules can be administered within
formulations that include one or more additional components, such
as a pharmaceutically acceptable carrier, diluent, excipient,
adjuvant, emulsifier, buffer, stabilizer, or preservative.
[0309] As used herein, the term "carrier" means a pharmaceutically
acceptable solid or liquid filler, diluent or encapsulating
material. A water-containing liquid carrier can contain
pharmaceutically acceptable additives such as acidifying agents,
alkalizing agents, antimicrobial preservatives, antioxidants,
buffering agents, chelating agents, complexing agents, solubilizing
agents, humectants, solvents, suspending and/or
viscosity-increasing agents, tonicity agents, wetting agents or
other biocompatible materials. Examples of ingredients of the above
categories can be found in the U.S. Pharmacopeia National
Formulary, 1990, pp. 1857-1859, as well as in Raymond C. Rowe, et
al., Handbook of Pharmaceutical Excipients, 5th ed., 2006, and
"Remington: The Science and Practice of Pharmacy," 21st ed., 2006,
editor David B. Troy.
[0310] Examples of preservatives include phenol, methyl paraben,
paraben, m-cresol, thiomersal, benzylalkonium chloride, and
mixtures thereof.
[0311] Examples of surfactants include oleic acid, sorbitan
trioleate, polysorbates, lecithin, phosphotidylcholines, various
long chain diglycerides and phospholipids, and mixtures
thereof.
[0312] Examples of phospholipids include phosphatidylcholine,
lecithin, phosphatidylglycerol, phosphatidylinositol,
phosphatidylserine, and phosphatidylethanolamine, and mixtures
thereof.
[0313] Examples of dispersants include ethylenediaminetetraacetic
acid.
[0314] Examples of gases include nitrogen, helium,
chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon
dioxide, air, and mixtures thereof.
[0315] In certain embodiments, the siNA and/or the polypeptide can
be encapsulated in liposomes, or reside either internal or external
to a liposome, or exist within liposome layers, or be administered
by iontophoresis, or incorporated into other vehicles, such as
hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive
microspheres, or proteinaceous vectors. See, for example, O'Hare
and Normand, International Publication No. WO/2000/53722.
Alternatively, a nucleic acid composition can be locally delivered
by direct injection or by use of an infusion pump. Direct injection
of the nucleic acid molecules of the invention, whether
subcutaneous, intramuscular, or intradermal, can take place using
standard needle and syringe methodologies, or by needle-free
technologies such as those described in Conry et al., Clin. Cancer
Res. 5:2330-2337, 1999, and Barry et al., International Publication
No. WO/1999/31262.
[0316] The compositions of this invention can be effectively
employed as pharmaceutical agents. Pharmaceutical agents prevent,
modulate the occurrence or severity of, or treat (alleviate one or
more symptom(s) to a detectable or measurable extent) of a disease
state or other adverse condition in a patient.
[0317] In some embodiments, this invention provides pharmaceutical
compositions and methods featuring the presence or administration
of one or more polynucleic acid(s), typically one or more siNAs,
combined, complexed, or conjugated with a lipid, which may further
be formulated with a pharmaceutically-acceptable carrier, such as a
diluent, stabilizer, or buffer.
[0318] Typically, the siNA will target a gene that is expressed at
an elevated level as a causal or contributing factor associated
with the subject disease state or adverse condition. In this
context, the siNA will effectively downregulate expression of the
gene to levels that prevent, alleviate, or reduce the severity or
recurrence of one or more associated disease symptoms.
Alternatively, for various distinct disease models where expression
of the target gene is not necessarily elevated as a consequence or
sequel of disease or other adverse condition, down regulation of
the target gene will nonetheless result in a therapeutic result by
lowering gene expression (i.e., to reduce levels of a selected mRNA
and/or protein product of the target gene). Alternatively, siNAs of
the invention may be targeted to lower expression of one gene,
which can result in upregulation of a "downstream" gene whose
expression is negatively regulated by a product or activity of the
target gene.
[0319] This siNAs of the present invention may be administered in
any form, for example transdermally or by local injection (e.g.,
local injection at sites of psoriatic plaques to treat psoriasis,
or into the joints of patients afflicted with psoriatic arthritis
or RA). In more detailed embodiments, the invention provides
formulations and methods to administer therapeutically effective
amounts of siNAs directed against of a mRNA of TNF-.alpha., which
effectively down-regulate the TNF-.alpha. RNA and thereby reduce or
prevent one or more TNF-.alpha.-associated inflammatory
condition(s). Comparable methods and compositions are provided that
target expression of one or more different genes associated with a
selected disease condition in animal subjects, including any of a
large number of genes whose expression is known to be aberrantly
increased as a causal or contributing factor associated with the
selected disease condition.
[0320] The compositions of the present invention may also be
formulated and used as tablets, capsules or elixirs for oral
administration, suppositories for rectal administration, sterile
solutions, suspensions for injectable administration, and the other
forms known in the art.
[0321] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
for example, systemic administration, into a cell or patient,
including for example a human. Suitable forms, in part, depend upon
the use or the route of entry, for example oral, transdermal,
transepithelial, or by injection. Such forms should not prevent the
composition or formulation from reaching a target cell (i.e., a
cell to which the negatively charged nucleic acid is desirable for
delivery). For example, pharmacological compositions injected into
the blood stream should be soluble. Other factors are known in the
art, and include considerations such as toxicity.
[0322] By "systemic administration" is meant in vivo systemic
absorption or accumulation of drugs in the blood stream followed by
distribution throughout the entire body. Administration routes
which lead to systemic absorption include, without limitation:
intravenous, subcutaneous, intraperitoneal, inhalation, oral,
intrapulmonary and intramuscular.
[0323] Examples of agents suitable for formulation with the nucleic
acid molecules of this invention include: P-glycoprotein inhibitors
(such as Pluronic P85), which can enhance entry of drugs into the
CNS (Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol.
13:16-26, 1999); biodegradable polymers, such as poly
(DL-lactide-coglycolide) microspheres for sustained release
delivery after intracerebral implantation (Emerich, D. F., et al.,
Cell Transplant 8:47-58, 1999, Alkermes, Inc., Cambridge, Mass.);
and loaded nanoparticles, such as those made of
polybutylcyanoacrylate, which can deliver drugs across the blood
brain barrier and can alter neuronal uptake mechanisms (Prog.
Neuropsychopharmacol Biol. Psychiatry 23:941-949, 1999). Other
examples of delivery strategies for the nucleic acid molecules of
the instant invention include material described in Boado, et al.,
J. Pharm. Sci. 87:1308-1315, 1998; Tyler, et al., FEBS Lett.
421:280-284, 1999; Pardridge, et al., PNAS USA. 92:5592-5596, 1995;
Boado, Adv. Drug Delivery Rev. 15:73-107, 1995; Aldrian-Herrada et
al., Nucleic Acids Res. 26:4910-4916, 1998; and Tyler, et al., PNAS
USA. 96:7053-7058, 1999.
[0324] The present invention also includes compositions prepared
for storage or administration, which include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers or diluents for
therapeutic use are well known in the pharmaceutical art, and are
described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing Co. (A. R. Gennaro ed. 1985). For example,
preservatives, stabilizers, dyes and flavoring agents may be
provided. These include sodium benzoate, sorbic acid and esters of
p-hydroxybenzoic acid. In addition, antioxidants and suspending
agents may be used.
[0325] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence of, treat, or alleviate a symptom
to some extent of a disease state. An amount of from 0.01 mg/kg to
50 mg/kg body weight/day of active nucleic acid should be
administered.
[0326] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents can be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0327] Oily suspensions can be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions can contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents can be added to provide palatable oral
preparations. These compositions can be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0328] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Additional
excipients, for example sweetening, flavoring and coloring agents,
can also be present.
[0329] Pharmaceutical compositions of the invention can also be in
the form of oil-in-water emulsions. The oily phase can be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents can be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0330] The pharmaceutical compositions can be in the form of a
sterile injectable aqueous or oleaginous suspension. This
suspension can be formulated according to the known art using those
suitable dispersing or wetting agents and suspending agents that
have been mentioned above. The sterile injectable preparation can
also be a sterile injectable solution or suspension in a non-toxic
parentally acceptable diluent or solvent, for example as a solution
in 1,3-butanediol. Among the acceptable vehicles and solvents that
can be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil can be employed including synthetic
mono-or diglycerides. In addition, fatty acids such as oleic acid
find use in the preparation of injectables.
[0331] The siNAs can also be administered in the form of
suppositories, for example, for rectal administration of the drug.
These compositions can be prepared by mixing the drug with a
suitable non-irritating excipient that is solid at ordinary
temperatures but liquid at the rectal temperature and will
therefore melt in the rectum to release the drug. Such materials
include cocoa butter and polyethylene glycols.
[0332] The siNAs can be modified extensively to enhance stability
by modification with nuclease resistant groups, for example,
2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H. For a review
see Usman and Cedergren, TIBS 17:34, 1992; Usman, et al., Nucleic
Acids Symp. Ser. 31:163, 1994. SiNA constructs can be purified by
gel electrophoresis using general methods or can be purified by
high pressure liquid chromatography and re-suspended in water.
[0333] Chemically synthesizing nucleic acid molecules with
modifications (base, sugar and/or phosphate) can prevent their
degradation by serum ribonucleases, which can increase their
potency. See for example, Eckstein, et al., International
Publication No. WO/1992/07065; Perrault et al., Nature 344:565,
1990; Pieken, et al., Science 253, 314, 1991; Usman and Cedergren,
Trends in Biochem. Sci. 17:334, 1992; Usman, et al., International
Publication No. WO/1993/15187; and Rossi et al., International
Publication No. WO/1991/03162; Sproat, U.S. Pat. No. 5,334,711; and
Gold, et al., U.S. Pat. No. 6,300,074. All of the above references
describe various chemical modifications that can be made to the
base, phosphate and/or sugar moieties of the nucleic acid molecules
described herein.
[0334] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-.beta.-allyl, 2'-H,
nucleotide base modifications. For a review, see Usman and
Cedergren, TIBS 17:34, 1992; Usman, et al., Nucleic Acids Symp.
Ser. 31:163, 1994; Burgin, et al., Biochemistry 35:14090, 1996.
Sugar modification of nucleic acid molecules have been extensively
described in the art. See Eckstein et al., International
Publication No. WO/1992/07065; Perrault, et al. Nature 344:565-568,
1990; Pieken, et al. Science 253:314-317, 1991; Usman and
Cedergren, Trends in Biochem. Sci. 17:334-339, 1992; Usman et al.
International Publication No. WO/1993/15187; Sproat, U.S. Pat. No.
5,334,711 and Beigelman, et al., J. Biol. Chem. 270:25702, 1995;
Beigelman, et al., International Publication No. WO/1997/26270;
Beigelman, et al., U.S. Pat. No. 5,716,824; Usman, et al., U.S.
Pat. No. 5,627,053; Woolf, et al., International Publication No.
WO/1998/13526; Thompson, et al., Karpeisky, et al., Tetrahedron
Lett. 39:1131, 1998; Earnshaw and Gait, Biopolymers (Nucleic Acid
Sciences) 48:39-55, 1998; Verma and Eckstein, Annu. Rev. Biochem.
67:99-134, 1998; and Burlina, et al., Bioorg. Med. Chem.
5:1999-2010, 1997. Such publications describe general methods and
strategies to determine the location of incorporation of sugar,
base and/or phosphate modifications and the like into nucleic acid
molecules without modulating catalysis. In view of such teachings,
similar modifications can be used as described herein to modify the
siNA nucleic acid molecules of the instant invention so long as the
ability of siNA to promote RNAi in cells is not significantly
inhibited.
[0335] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorodithioate,
and/or 5'-methylphosphonate linkages improves stability, excessive
modifications can cause some toxicity or decreased activity.
Therefore, when designing nucleic acid molecules, the amount of
these internucleotide linkages should be minimized. The reduction
in the concentration of these linkages should lower toxicity,
resulting in increased efficacy and higher specificity of these
molecules.
[0336] In one embodiment, the invention features modified siNA
molecules, with phosphate backbone modifications comprising one or
more phosphorothioate, phosphorodithioate, methylphosphonate,
phosphotriester, morpholino, amidate carbamate, carboxymethyl,
acetamidate, polyamide, sulfonate, sulfonamide, sulfamate,
formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a
review of oligonucleotide backbone modifications, see Hunziker and
Leumann, Nucleic Acid Analogues: Synthesis and Properties, in
Modern Synthetic Methods, VCH, 1995, pp. 331-417, and Mesmaeker, et
al., "Novel Backbone Replacements for Oligonucleotides, in
Carbohydrate Modifications in Antisense Research," ACS, 1994, pp.
24-39.
[0337] Methods for the delivery of nucleic acid molecules are
described in Akhtar, et al., Trends Cell Bio. 2:139, 1992;
"Delivery Strategies for Antisense Oligonucleotide Therapeutics,"
ed. Akhtar, 1995; Maurer, et al., Mol. Membr. Biol. 16:129-140,
1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165-192, 1999;
and Lee, et al., ACS Symp. Ser. 752:184-192, 2000. Beigelman, et
al., U.S. Pat. No. 6,395,713, and Sullivan et al., International
Publication No. WO/1994/02595 further describe the general methods
for delivery of nucleic acid molecules. These protocols can be
utilized for the delivery of virtually any nucleic acid molecule.
Nucleic acid molecules can be administered to cells by a variety of
methods known to those of skill in the art, including, but not
restricted to, encapsulation internally or externally by liposomes,
by iontophoresis, or by incorporation into other vehicles, such as
biodegradable polymers, hydrogels, cyclodextrins (see e.g.
Gonzalez, et al., Bioconjugate Chem. 10:1068-1074, 1999; Wang, et
al., International Publication Nos. WO/2003/47518 and
WO/2003/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA
microspheres (see e.g. U.S. Pat. No. 6,447,796 and U.S. Patent
Application Publication No. US 2002/130430), biodegradable
nanocapsules, and bioadhesive microspheres, or by proteinaceous
vectors (O'Hare and Normand, International Publication No.
WO/2000/53722). Alternatively, the nucleic acid/vehicle combination
is locally delivered by direct injection or by use of an infusion
pump. Direct injection of the nucleic acid molecules of the
invention, whether subcutaneous, intramuscular, or intradermal, can
take place using standard needle and syringe methodologies, or by
needle-free technologies such as those described in Conry, et al.,
Clin. Cancer Res. 5:2330-2337, 1999, and Barry, et al.,
International Publication No. WO/1999/31262. The molecules of the
instant invention can be used as pharmaceutical agents.
Pharmaceutical agents prevent, modulate the occurrence, or treat
(alleviate a symptom to some extent, preferably all of the
symptoms) of a disease state in a subject.
[0338] By "RNA" is meant a molecule comprising at least one
ribonucleotide residue. By "ribonucleotide" is meant a nucleotide
with a hydroxyl group at the 2' position of a
.beta.-D-ribo-furanose moiety. The terms include double-stranded
RNA, single-stranded RNA, isolated RNA such as partially purified
RNA, essentially pure RNA, synthetic RNA, recombinantly produced
RNA, as well as altered RNA that differs from naturally occurring
RNA by the addition, deletion, substitution and/or alteration of
one or more nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of the siNA or
internally, for example, at one or more nucleotides of the RNA.
Nucleotides in the RNA molecules of the instant invention can also
comprise non-standard nucleotides, such as non-naturally occurring
nucleotides or chemically synthesized nucleotides or
deoxynucleotides. These altered RNAs can be referred to as analogs
or analogs of naturally-occurring RNA.
[0339] By "cap structure" is meant chemical modifications, which
have been incorporated at either terminus of the oligonucleotide
(see, e.g. Adamic, et al., U.S. Pat. No. 5,998,203, incorporated by
reference herein). These terminal modifications protect the nucleic
acid molecule from exonuclease degradation, and may help in
delivery and/or localization within a cell. The cap may be present
at the 5'-terminus (5'-cap) or at the 3'-terminal (3'-cap) or may
be present on both termini. In non-limiting examples, the 5'-cap
includes, but is not limited to, glyceryl, inverted deoxy abasic
residue (moiety); 4',5'-methylene nucleotide;
1-(.beta.-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety.
[0340] Examples of the 3'-cap include, but are not limited to,
glyceryl, inverted deoxy abasic residue (moiety), 4',5'-methylene
nucleotide; 1-(.beta.-D-erythrofuranosyl) nucleotide; 4'-thio
nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate;
1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and Lyer, Tetrahedron 49:1925, 1993;
incorporated by reference herein).
[0341] By the term "non-nucleotide" is meant any group or compound
which can be incorporated into a nucleic acid chain in the place of
one or more nucleotide units, including either sugar and/or
phosphate substitutions, and allows the remaining bases to exhibit
their enzymatic activity. The group or compound is abasic in that
it does not contain a commonly recognized nucleotide base, such as
adenosine, guanine, cytosine, uracil or thymine and therefore lacks
a base at the 1'-position.
[0342] By "nucleotide" as used herein is as recognized in the art
to include natural bases (standard), and modified bases well known
in the art. Such bases are generally located at the 1' position of
a nucleotide sugar moiety. Nucleotides generally comprise a base,
sugar and a phosphate group. The nucleotides can be unmodified or
modified at the sugar, phosphate and/or base moiety, (also referred
to interchangeably as nucleotide analogs, modified nucleotides,
non-natural nucleotides, non-standard nucleotides and other; see,
e.g. Usman and McSwiggen, supra; Eckstein, et al., International
Publication No. WO/1992/07065; Usman, et al, International
Publication No. WO/1993/15187; Uhlman & Peyman, supra, all are
hereby incorporated by reference herein). There are several
examples of modified nucleic acid bases known in the art as
summarized by Limbach, et al., Nucleic Acids Res. 22:2183, 1994.
Some of the non-limiting examples of base modifications that can be
introduced into nucleic acid molecules include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,
naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),
5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
6-methyluridine), propyne, and others (Burgin, et al., Biochemistry
35:14090, 1996; Uhlman & Peyman, supra). By "modified bases" in
this aspect is meant nucleotide bases other than adenine, guanine,
cytosine and uracil at 1' position or their equivalents.
[0343] By "target site" or "target sequence" or "targeted sequence"
is meant a sequence within a target nucleic acid (e.g., RNA) that
is "targeted" for cleavage mediated by a siNA construct which
contains sequences within its antisense region that are
complementary to the target sequence.
[0344] The siNA molecules can be complexed with cationic lipids,
packaged within liposomes, or otherwise delivered to target cells
or tissues. The nucleic acid or nucleic acid complexes can be
locally administered to through injection, infusion pump or stent,
with or without their incorporation in biopolymers. In another
embodiment, polyethylene glycol (PEG) can be covalently attached to
siNA compounds of the present invention, to the polypeptide, or
both. The attached PEG can be any molecular weight, preferably from
about 2,000 to about 50,000 daltons (Da).
[0345] The sense region can be connected to the antisense region
via a linker molecule, such as a polynucleotide linker or a
non-nucleotide linker.
[0346] "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.
[0347] "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).
[0348] "Large double-stranded RNA" refers to any double-stranded
RNA having a size greater than about 40 by for example, larger than
100 by 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.
[0349] 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.
[0350] "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.
[0351] "One or more dsRNAs" refers to dsRNAs that differ from each
other on the basis of primary sequence.
[0352] "Target gene or mRNA" refers to any gene or mRNA of
interest. Indeed any of the genes previously identified by genetics
or by sequencing may represent a target. Target genes or mRNA may
include developmental genes and regulatory genes as well as
metabolic or structural genes or genes encoding enzymes. The target
gene may be expressed in those 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.
[0353] All publications, references, patents, patent publications
and patent applications cited herein are each hereby specifically
incorporated by reference in entirety.
[0354] While this invention has been described in relation to
certain embodiments, and many details have been set forth for
purposes of illustration, it will be apparent to those skilled in
the art that this invention includes additional embodiments, and
that some of the details described herein may be varied
considerably without departing from this invention. This invention
includes such additional embodiments, modifications and
equivalents. In particular, this invention includes any combination
of the features, terms, or elements of the various illustrative
components and examples.
[0355] The use herein of the terms "a," "an," "the" and similar
terms in describing the invention, and in the claims, are to be
construed to include both the singular and the plural. The terms
"comprising," "having," "including" and "containing" are to be
construed as open-ended terms which mean, for example, "including,
but not limited to." Recitation of a range of values herein refers
individually to each and any separate value falling within the
range as if it were individually recited herein, whether or not
some of the values within the range are expressly recited. For
example, the range "4 to 12" includes without limitation the values
5, 5.1, 5.35 and any other whole, integer, fractional, or rational
value greater than or equal to 4 and less than or equal to 12.
Specific values employed herein will be understood as exemplary and
not to limit the scope of the invention.
[0356] Definitions of technical terms provided herein should be
construed to include without recitation those meanings associated
with these terms known to those skilled in the art, and are not
intended to limit the scope of the invention. Definitions of
technical terms provided herein shall be construed to dominate over
alternative definitions in the art or definitions which become
incorporated herein by reference to the extent that the alternative
definitions conflict with the definition provided herein.
[0357] The examples given herein, and the exemplary language used
herein are solely for the purpose of illustration, and are not
intended to limit the scope of the invention.
[0358] When a list of examples is given, such as a list of
compounds or molecules suitable for this invention, it will be
apparent to those skilled in the art that mixtures of the listed
compounds or molecules are also suitable.
EXAMPLE 1
In Vitro Assay for LacZ Gene Expression Knockdown in 9L Cells
[0359] 9L/LacZ is a rat gliosarcoma cell line stably expressing the
LacZ gene that encodes bacterial galactosidase. LacZ gene knockdown
measurements can be used as a primary activity-based in vitro assay
for interfering RNA delivery formulations.
[0360] For LacZ gene knockdown measurements, 9L/LacZ cells were
transfected with an RNAi formulation, and a .beta.-galactosidase
assay was performed on cells harvested at day 3 post transfection.
An additional assay was performed to quantify protein
concentration.
[0361] 9L/LacZ cells were plated at 8000 cells/well (96-well) and
incubated overnight in medium. Confluency was about 15-20% at the
time of transfection. Transfection complex was prepared by adding
an interfering RNA to OptiMEM.TM. medium and vortexing, separately
adding a delivery formulation to OptiMEM.TM. medium and vortexing,
and finally mixing the interfering RNA in medium with the delivery
formulation in medium to make the transfection complex. The medium
for incubated cells was replaced with fresh no-serum media
(OptiMEM.TM. without serum) and transfection complex was added to
each well. Cells were incubated 5 hrs, then after the addition of
100 microliters complete medium (DMEM plus 10% fetal bovine serum)
were incubated overnight at 37.degree. C. and 5% CO.sub.2. The next
day, 24 hours after transfection, the medium was changed to fresh
complete medium and the cells were incubated another 48 hrs at
37.degree. C. and 5% CO.sub.2.
[0362] For LacZ gene knockdown, the harvested 9L/LacZ cells were
washed in PBS, lysed in M-PER.TM. Reagent (Pierce), and incubated
at room temperature for 15 minutes. Lysate was taken from each well
for protein assay with a Micro BCA kit (Pierce, Thermo Fisher
Scientific) and .beta.-gal assay with All-in-One.TM.
.beta.-Galactosidase Assay Reagent (Pierce).
In Vitro Assay for PPIB Gene Expression Knockdown in A549 cells
[0363] Cyclophilin B (PPIB) gene knockdown measurements can be used
as a primary activity-based in vitro assay for interfering RNA
delivery formulations. Cyclophilin B (PPIB) gene expression
knockdown was measured in A549 human alveolar basal epithelial
cells. For PPIB gene knockdown measurements, A549 cells were
transfected with an interfering RNA formulation, total RNA prepared
24 hours after transfection, and PPIB mRNA assayed by RT-PCR.
QRT-PCR of 36B4 (acidic ribosomal phosphoprotein PO) mRNA
expression was performed for normalization.
[0364] A549 cells were seeded at 7,500 cells/well (96-well) and
incubated overnight in medium. Confluency was about 50% at the time
of transfection. Transfection complex was prepared by adding an
interfering RNA to medium (OptiMEM.TM.) and vortexing, separately
adding a delivery formulation to medium (OptiMEM.TM.) and
vortexing, and finally mixing the interfering RNA in medium with
the delivery formulation in medium and incubating 20 minutes at
room temperature to make the transfection complex. The medium for
incubated cells was replaced with fresh OptiMEM.TM. and
transfection complex was added to each well. Cells were incubated
for 5 hrs at 37.degree. C. and 5% CO.sub.2, then complete medium
was added (to a final fetal bovine serum concentration 10%) and
incubation continued until 24 hours post-transfection.
[0365] For PPIB gene knockdown cells were lysed and RNA prepared
(Invisorb RNA Cell HTS 96-Kit/C, Invitek, Berlin, or RNeasy 96 Kit,
Qiagen). Quantitative RT-PCR was performed using One-Step qRT-PCR
kit (Invitrogen) on a DNA Engine Opticon2 thermal cycler
(BioRad).
TABLE-US-00009 Primers used for PPIB were: (SEQ ID NO: 323)
5'-GGCTCCCAGTTCTTCATCAC-3' (forward) and (SEQ ID NO: 324)
5'-CCTTCCGCACCACCTC-3' (reverse) with (SEQ ID NO: 325)
5'-FAM-CTAGATGGCAAGCATGTGGTGTTTGG- TAMRA-3' for the probe. For
36B4, primers were: (SEQ ID NO: 326) 5'-TCTATCATCAACGGGTACAAACGA-3'
(forward) and (SEQ ID NO: 327) 5'-CTTTTCAGCAAGTGGGAAGGTG-3'
(reverse) with (SEQ ID NO: 328) 5'-FAM-CCTGGCCTTGTCTGTGGAGACGGATTA-
TAMRA-3' for the probe.
In Vivo Assay for Influenza Viral Titer Knockdown in Mouse
[0366] Influenza viral titer knockdown measurements in mice can be
used as an in vivo gauge of efficacy for interfering RNA
lipopeptide delivery formulations.
[0367] In this assay, typically 50 uL of a interfering RNA
lipopeptide formulation, or PBS for a control group, was
administered intranasally in 7-9 week old Balb/C mice anesthetized
with ketamine/xylazine. Daily dosing was performed for 3
consecutive days on days -2, -1, and 0. Infection was induced 4
hours after the last dosing.
[0368] Influenza infection was induced with Influenza A/Puerto
Rico/8/34 (PR8, subtype H1N1). For infection, 50 .mu.l of 20 pfu
PR8 diluted in 0.3% BSA/1XPBS/PS was administered intranasally into
mice anesthetized with ketamine/xylazine. 48 hours after infection,
the lungs were harvested and homogenized in 600 uL 0.3%
BSA/1XPBS/PS. The homogenates were frozen and thawed twice to
release the virus. A TCID50 assay (Tissue-Culture Infectious Dose
50) was performed to titer virus in lung homogenates. Flat-bottom,
96-well plates were seeded with 2.times.10.sup.4 MDCK cells per
well, and 24 hours later, the serum-containing medium was removed.
30 uL of lung homogenates, either undiluted or diluted from 10- to
10.sup.7-fold (in 10-fold steps), was added into quadruplicate
wells. After incubation for 1 hr, 170 .mu.l of infection medium
(DMEM/0.3% BSA/10 mM HEPES/PS) containing 4 .mu.g/ml trypsin was
added to each well. After incubation for 48 hours at 37.degree. C.,
the presence or absence of virus in the culture supernatants was
determined by hemagglutination of chicken red blood cells. The
virus titers were estimated using the Spearman and Karber
formula.
SYBR.TM. Gold Assay for siRNA Concentrations
[0369] The concentration of dsRNA in a formulation can be
determined by SYBR.TM. Gold assay as follows: 10 ul of dsRNA
formulation is added to 100 ul MeOH and incubated for 5 minutes at
55.degree. C. 50 ul Heparin (200 mg/ml) is added, and the solution
is incubated for 5 minutes at 55.degree. C. 790 ul PBS (phosphate
buffered saline) is added, and the sample is spun down in microfuge
to pelletize. A 90 ul sample of the pellet is incubated with 10 ul
SYBR.TM. Gold reagent (Molecular Probes, Eugene, Oreg.).
Fluorescence is read at Em 535 nm with excitation at 495 nm.
EXAMPLE 2
[0370] The structure of some double-stranded RNAs of this
disclosure are shown in Table 6.
TABLE-US-00010 TABLE 6 Double-stranded RNAs DX3030 (SEQ ID NO: 329)
Influenza Sense 5'-GGAUCUUAUUUCUUCGGAGACAAdTdG-3' (SEQ ID NO: 330)
Antisense 5'-CAUUGUCUCCGAAGAAAUAAGAUCCUU- 3' DX2816 (SEQ ID NO:
331) Non- Sense 5'-UUCUCCGAACGUGUCACGUdTdT-3' target (SEQ ID NO:
332) Qneg Antisense 5'-ACGUGACACGUUCGGAGAAdTdT-3' DX2940 (SEQ ID
NO: 333) LacZ Sense 5'-CUACACAAAUCAGCGAUUUdTdT-3' (SEQ ID NO: 334)
Antisense 5'-AAAUCGCUGAUUUGUGUAGdTdC-3' DX2742 (SEQ ID NO: 335)
PPIB Sense 5'-GGAAAGACUGUUCCAAAAAUU-3' MoCypB (SEQ ID NO: 336)
Antisense 5'-UUUUUGGAACAGUCUUUCCUU-3' DX 2744 (SEQ ID NO: 337)
G1498 Sense 5'-GGAUCUUAUUUCUUCGGAGdTdT-3' influenza (SEQ ID NO:
338) Antisense 5'-CUCCGAAGAAAUAAGAUCCdTdT-3' DX 2918 (SEQ ID NO:
339) Inm4 Sense 5'-CCGTCAGCCGATTTGCTATTT-3' TNFa (SEQ ID NO: 340)
modified Antisense 5'-p-AUAGCAAATCGGCTGACGGTT-3'
EXAMPLE 3
[0371] Active RNA formulations of this disclosure can be prepared
by dissolving an interfering RNA in buffer or cell culture medium
and vortexing, separately admixing a delivery formulation with
buffer or cell culture medium and vortexing, and finally admixing
the interfering RNA mixture with the delivery formulation mixture
to make an active RNAi transfection formulation.
[0372] To prepare a delivery formulation, lipopeptides along with
other lipids and/or excipients can be solubilized in
CHCl.sub.3/MeOH, dried down under N.sub.2, and hydrated in 10 mM
HEPES with 5% dextrose at pH 7.4. The mixture can be sonicated, or
extruded, dialyzed, and/or tangential flow filtered.
[0373] An exemplary interfering RNA formulation of this disclosure
is shown in Table 7. In this example, the lipopeptide provides its
own formulation for intracellular delivery of an interfering siRNA
therapeutic. The amount of lipopeptide is given as the mole
percentage of delivery components, not including the active RNA
agent.
TABLE-US-00011 TABLE 7 siRNA Formulation Component Amount siRNA 50
nM lipopeptide 100 mole %
[0374] An exemplary interfering RNA formulation of this disclosure
is shown in Table 8. In this example, the lipopeptide provides its
own formulation for intracellular delivery of an interfering siRNA
therapeutic. The amount of lipopeptide is given as the mole
percentage of delivery components, not including the active RNA
agent. The length of the lipophilic group for each peptide on the
proceeding tables is represented by "(CX)" where "X" represents the
number of carbons in the lipophilic group. For example, C20
represents a lipophilic group with a 20 carbon chain length.
TABLE-US-00012 TABLE 8 dsRNA Formulation Component Amount dsRNA 50
nM (C16)--(H).sub.8(R).sub.8(H).sub.8K--NH--(C16) 100 mole % (SEQ
ID NO: 13)
[0375] An exemplary RNAi formulation of this disclosure is shown in
Table 9. In this example, the lipopeptide is combined with a
non-cationic lipid in a co-delivery formulation.
TABLE-US-00013 TABLE 9 DX3030 Formulation Component Amount siRNA
DX3030 50 nM (C20)--(H).sub.8(R).sub.8(H).sub.8K--NH--(C20) 50 mole
% (SEQ ID NO: 13) 1,2-Distearoyl-sn-glycero-3- 50 mole %
phosphoethanolamine (DSPE)
[0376] An exemplary RNAi formulation of this disclosure is shown in
Table 10. In this example, a lipopeptide is combined with a
cationic lipid, a non-cationic lipid, and a pegylated lipid in a
multicomponent delivery formulation.
TABLE-US-00014 TABLE 10 dsRNA Formulation Component Amount dsRNA 25
nM (C20)--(H).sub.8(R).sub.8(H).sub.8K--NH--(C20) 50 mole % (SEQ ID
NO: 13) DSPC 49 mole % DSPE-PEG2000 1 mole %
[0377] Examples of additional lipids for RNAi formulations of this
disclosure are shown in Table 11.
TABLE-US-00015 TABLE 11 Lipids for Delivery Formulations Material
MW DOTAP Avanti 890890C 698.55 DOPE Avanti 850725C 744.04 DPhyPE
Avanti 850402P 804.18
EXAMPLE 4
[0378] The knockdown activities of example formulations of
interfering RNA compositions of this disclosure are shown in Table
12.
TABLE-US-00016 TABLE 12 Knockdown for Delivery Formulations SEQ %
KD (vs % KD N:P ID QNeg) (vsQNeg) Ratio NO: Lipopeptide A549/PPIB
9L/lacZ 2 -- RNAiMAX .TM. 73.4 82.4 2 350
(C18)-HHHHRRRRRRRR-Cysteamide 15.3 -0.8 2 351
(C18)-PEG27-HHHHHHHHRRRRRRRR-amide 30.6 9.2 2 352
Ac-GALFLAFLAAALSLMGLWSQPKKKRKV-Cysteamide 20.5 29.1 2 353
Ac-GALFLAFLAAALSLMGLWSQPKSKRKV-Cysteamide 15.5 29.5 2 296
Ac-LIRLWSHLIHIWFQNRRLKWKKK-amide 8.7 -19.9 2 13
(C20)--(H).sub.8(R).sub.8(H).sub.8K--NH--(C20) 29.7 60.1 2 354
(C16)-HHHHHKHHHKKKHKHKKK-Cysteamide 14.7 12.0 2 355
(C18)-HHHHHKHHHKKKHKHKKK-cysteamide 25.5 -11.3 2 356
Ac-GALFLGFLGAAG STMGAWSQPKSKRKV-amide -8.1 -0.4 4 10
(C18)-PEG27-HHHHHHHHRRRRRRRR-amide 7.4 -16.1 4 353
Ac-GALFLAFLAAALSLMGLWSQPKSKRKV-Cysteamide 16.0 -9.3 4 296
Ac-LIRLWSHLIHIWFQNRRLKWKKK-amide -28.8 -4.5 4 13
(C20)--(H).sub.8(R).sub.8(H).sub.8K--NH--(C20) 65.8 57.5 4 354
(C16)-HHHHHKHHHKKKHKHKKK-Cysteamide -19.8 12.4 4 355
(C18)-HHHHHKHHHKKKHKHKKK-cysteamide 21.6 -19.4 4 356
Ac-GALFLGFLGAAG STMGAWSQPKSKRKV-amide -3.7 -1.6
[0379] For the lipopeptides in Table 12, the results showed that at
least two lipopeptides, (C20)-(H).sub.8(R).sub.8(H).sub.8K-NH-(C20)
(SEQ ID NO: 13) and (C18)-HHHHHKHHHKKKHKHKKK-cysteamide (SEQ ID NO:
355), exhibited significant knockdown in at least the A549/PPIB
assay as compared to both Qneg and vehicle (data for vehicle not
shown).
Sequence CWU 1
1
356120PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 1Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg 20220PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 2Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10
15Lys Lys Lys Lys 20340PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 3Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 30Lys Lys Lys Lys Lys
Lys Lys Lys 35 40440PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 4Arg His Arg His Arg His Arg His Arg His
Arg His Arg His Arg His1 5 10 15Arg His Arg His Arg His Arg His Arg
His Arg His Arg His Arg His 20 25 30Arg His Arg His Arg His Arg His
35 40580PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 5Arg His Arg His Arg His Arg His Arg His Arg His
Arg His Arg His1 5 10 15Arg His Arg His Arg His Arg His Arg His Arg
His Arg His Arg His 20 25 30Arg His Arg His Arg His Arg His His Arg
His Arg His Arg His Arg 35 40 45His Arg His Arg His Arg His Arg His
Arg His Arg His Arg His Arg 50 55 60His Arg His Arg His Arg His Arg
His Arg His Arg His Arg His Arg65 70 75 80640PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 6Lys
His Lys His Lys His Lys His Lys His Lys His Lys His Lys His1 5 10
15Lys His Lys His Lys His Lys His Lys His Lys His Lys His Lys His
20 25 30Lys His Lys His Lys His Lys His 35 40780PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 7Lys
His Lys His Lys His Lys His Lys His Lys His Lys His Lys His1 5 10
15Lys His Lys His Lys His Lys His Lys His Lys His Lys His Lys His
20 25 30Lys His Lys His Lys His Lys His His Lys His Lys His Lys His
Lys 35 40 45His Lys His Lys His Lys His Lys His Lys His Lys His Lys
His Lys 50 55 60His Lys His Lys His Lys His Lys His Lys His Lys His
Lys His Lys65 70 75 80822PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 8His Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys His
20922PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 9His Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Arg His 201016PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 10His
His His His His His His His Arg Arg Arg Arg Arg Arg Arg Arg1 5 10
151112PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 11His His His His Arg Arg Arg Arg Arg Arg Arg
Arg1 5 101212PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 12His His His His Arg Arg Arg Arg Lys
Lys Lys Lys1 5 101325PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 13His His His His His His His
His Arg Arg Arg Arg Arg Arg Arg Arg1 5 10 15His His His His His His
His His Lys 20 251415PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 14His His His His Arg Arg Arg
Arg Arg His His His Lys Lys Lys1 5 10 151518PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 15His
His His His His Lys His His His Lys Lys Lys His Lys His Lys1 5 10
15Lys Lys1636PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 16Lys Gly Ser Lys Lys Ala Val Thr Lys
Ala Gln Lys Lys Asp Gly Lys1 5 10 15Lys Arg Lys Arg Ser Arg Lys Glu
Ser Tyr Ser Val Tyr Val Tyr Lys 20 25 30Val Leu Lys Gln
351736PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 17Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys
Lys Glu Gly Lys1 5 10 15Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser
Val Tyr Val Tyr Lys 20 25 30Val Leu Lys Gln 351827PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 18Ala
Gln Lys Lys Glu Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser1 5 10
15Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln 20
251918PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 19Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val
Tyr Lys Val Leu1 5 10 15Lys Gln2013PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 20Glu
Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln1 5
10216PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 21Tyr Lys Val Leu Lys Gln1 52215PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 22Arg
Val Ile Arg Trp Phe Gln Asn Lys Arg Ser Lys Asp Lys Lys1 5 10
152327PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 23Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly
Ser Thr Met Gly1 5 10 15Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val
20 252416PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 24Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met
Lys Trp Lys Lys1 5 10 152516PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 25Arg Gln Ile Lys Ile Trp Phe
Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 152626PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 26Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys1 5 10
15Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 252721PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 27Leu
Leu Asn Gln Leu Ala Gly Arg Met Ile Pro Lys Trp Ser Gln Lys1 5 10
15Ser Lys Arg Lys Val 202820PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 28Thr Leu Asp His Val Leu Asp
His Val Gln Thr Trp Ser Gln Lys Ser1 5 10 15Lys Arg Lys Val
202923PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 29Ser Tyr Phe Ile Leu Arg Arg Arg Arg Lys Arg Phe
Pro Tyr Phe Phe1 5 10 15Thr Asp Val Arg Val Ala Ala
203010PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 30Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg1 5
10318PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 31Arg Arg Arg Arg Arg Arg Arg Arg1
53228PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 32Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp
Ser Gln Pro Gly1 5 10 15Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro
Gln 20 253317PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 33Gly Arg Pro Arg Glu Ser Gly Lys Lys
Arg Lys Arg Lys Arg Leu Lys1 5 10 15Pro3413PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 34Lys
Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln1 5
103513PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 35Glu Ser Tyr Ser Val Tyr Val Tyr Arg Val Leu Arg
Gln1 5 103613PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 36Arg Ser Tyr Ser Val Tyr Val Tyr Arg
Val Leu Arg Gln1 5 103713PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 37Gln Lys Leu Val Lys Tyr Val
Tyr Val Ser Tyr Ser Glu1 5 103813PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 38Glu Ser Tyr Ser Val Tyr
Val Tyr Lys Val Leu Lys Gln1 5 103913PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 39Ala
Ser Tyr Ser Val Tyr Val Tyr Ala Val Leu Ala Gln1 5
104013PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 40Gln Lys Leu Val Lys Tyr Val Tyr Val Ser Tyr Ser
Glu1 5 104119PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 41Arg Arg Arg Arg Arg Arg Glu Ser Tyr
Ser Val Tyr Val Tyr Lys Val1 5 10 15Leu Lys Gln4219PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 42Glu
Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln Arg Arg Arg1 5 10
15Arg Arg Arg4322PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 43Arg Arg Arg Arg Arg Arg Arg Gln Ile
Lys Ile Trp Phe Gln Asn Arg1 5 10 15Arg Met Lys Trp Lys Lys
204422PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 44Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met
Lys Trp Lys Lys1 5 10 15Arg Arg Arg Arg Arg Arg 204517PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 45Lys
Thr Lys Ile Glu Ser Leu Lys Glu His Gly Arg Arg Arg Arg Arg1 5 10
15Arg4631PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 46Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val
Pro Ala Gln Asn1 5 10 15Ala Ile Ser Thr Thr Phe Pro Tyr Thr Arg Arg
Arg Arg Arg Arg 20 25 304726PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 47Gly Leu Phe Glu Ala Leu Leu
Glu Leu Leu Glu Ser Leu Trp Glu Leu1 5 10 15Leu Leu Glu Ala Arg Arg
Arg Arg Arg Arg 20 254818PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 48Leu Leu Asn Gln Leu Ala Gly
Arg Met Ile Pro Lys Arg Arg Arg Arg1 5 10 15Arg
Arg4917PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 49Thr Leu Asp His Val Leu Asp His Val Gln Thr Arg
Arg Arg Arg Arg1 5 10 15Arg5026PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 50Gly Leu Phe Gly Ala Ile Ala
Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile Asp Gly Arg Arg
Arg Arg Arg Arg 20 255117PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 51Lys Glu Thr Trp Trp Glu Thr
Trp Trp Thr Glu Arg Arg Arg Arg Arg1 5 10 15Arg5216PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 52His
His His His His His His His His His Arg Arg Arg Arg Arg Arg1 5 10
155322PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 53Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala
Leu Leu Ala Pro1 5 10 15Arg Arg Arg Arg Arg Arg 20545PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 54Lys
Val Leu Lys Gln1 5557PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 55Lys Arg Arg Gln Arg Arg
Arg1 55634PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 56Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala
Ser Arg Pro Thr1 5 10 15Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser
Arg Pro Arg Arg Pro 20 25 30Val Asp5715PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 57Val
Thr Val Leu Ala Leu Gly Ala Leu Ala Gly Val Gly Val Gly1 5 10
155817PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 58Gly Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly
Ser Thr Met Gly1 5 10 15Ala5917PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 59Met Gly Leu Gly Leu His Leu
Leu Val Leu Ala Ala Ala Leu Gln Gly1 5 10 15Ala6024PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 60Leu
Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln1 5 10
15Thr Ala Ile Gly Val Gly Ala Pro 206126PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 61Gly
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Ile Asn Leu Lys1 5 10
15Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 256216PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 62Thr
Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro Lys Lys Lys Lys1 5 10
156318PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 63Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys
Ala Ala Leu Lys1 5 10 15Leu Ala6416PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 64Gly
Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10
156516PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 65Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly
Val Ala Thr Ala1 5 10 156616PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 66Phe Leu Gly Phe Leu Leu Gly
Val Gly Ser Ala Ile Ala Ser Gly Val1 5 10 156716PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 67Gly
Val Phe Val Leu Gly Phe Leu Gly Phe Leu Ala Thr Ala Gly Ser1 5 10
156816PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 68Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro Tyr Phe
Gly Pro Ala Ala1 5 10 156956PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 69Ala Cys Thr Cys Pro Tyr Cys
Lys Asp Ser Glu Gly Arg Gly Ser Gly1 5 10 15Asp Pro Gly Lys Lys Lys
Gln His Ile Cys His Ile Gln Gly Cys Gly 20 25 30Lys Val Tyr Gly Lys
Thr Ser His Leu Arg Ala His Leu Arg Trp His 35 40 45Thr Gly Glu Arg
Pro Phe Met Cys 50 557054PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 70Ala Cys Thr Cys Pro Asn Cys
Lys Asp Gly Glu Lys Arg Ser Gly Glu1 5 10 15Gln Gly Lys Lys Lys His
Val Cys His Ile Pro Asp Cys Gly Lys Thr 20 25 30Phe Arg Lys Thr Ser
Leu Leu Arg Ala His Val Arg Leu His Thr Gly 35 40 45Glu Arg Pro Phe
Val Cys 507155PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 71Ala Cys Thr Cys Pro Asn Cys Lys Glu
Gly Gly Gly Arg Gly Thr Asn1 5 10 15Leu Gly Lys Lys Lys Gln His Ile
Cys His Ile Pro Gly Cys Gly Lys 20 25 30Val Tyr Gly Lys Thr Ser His
Leu Arg Ala His Leu Arg Trp His Ser 35 40 45Gly Glu Arg Pro Phe Val
Cys 50
557256PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 72Ala Cys Ser Cys Pro Asn Cys Arg Glu Gly Glu Gly
Arg Gly Ser Asn1 5 10 15Glu Pro Gly Lys Lys Lys Gln His Ile Cys His
Ile Glu Gly Cys Gly 20 25 30Lys Val Tyr Gly Lys Thr Ser His Leu Arg
Ala His Leu Arg Trp His 35 40 45Thr Gly Glu Arg Pro Phe Ile Cys 50
557360PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 73Arg Cys Thr Cys Pro Asn Cys Thr Asn Glu Met Ser
Gly Leu Pro Pro1 5 10 15Ile Val Gly Pro Asp Glu Arg Gly Arg Lys Gln
His Ile Cys His Ile 20 25 30Pro Gly Cys Glu Arg Leu Tyr Gly Lys Ala
Ser His Leu Lys Thr His 35 40 45Leu Arg Trp His Thr Gly Glu Arg Pro
Phe Leu Cys 50 55 607458PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 74Thr Cys Asp Cys Pro Asn Cys
Gln Glu Ala Glu Arg Leu Gly Pro Ala1 5 10 15Gly Val His Leu Arg Lys
Lys Asn Ile His Ser Cys His Ile Pro Gly 20 25 30Cys Gly Lys Val Tyr
Gly Lys Thr Ser His Leu Lys Ala His Leu Arg 35 40 45Trp His Thr Gly
Glu Arg Pro Phe Val Cys 50 557553PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 75Arg Cys Thr Cys Pro Asn
Cys Lys Ala Ile Lys His Gly Asp Arg Gly1 5 10 15Ser Gln His Thr His
Leu Cys Ser Val Pro Gly Cys Gly Lys Thr Tyr 20 25 30Lys Lys Thr Ser
His Leu Arg Ala His Leu Arg Lys His Thr Gly Asp 35 40 45Arg Pro Phe
Val Cys 507656PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 76Pro Gln Ile Ser Leu Lys Lys Lys Ile
Phe Phe Phe Ile Phe Ser Asn1 5 10 15Phe Arg Gly Asp Gly Lys Ser Arg
Ile His Ile Cys His Leu Cys Asn 20 25 30Lys Thr Tyr Gly Lys Thr Ser
His Leu Arg Ala His Leu Arg Gly His 35 40 45Ala Gly Asn Lys Pro Phe
Ala Cys 50 557731PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 77Trp Trp Glu Thr Trp Lys Pro Phe Gln
Cys Arg Ile Cys Met Arg Asn1 5 10 15Phe Ser Thr Arg Gln Ala Arg Arg
Asn His Arg Arg Arg His Arg 20 25 307816PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 78Gly
Lys Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu1 5 10
157916PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 79Arg Val Ile Arg Val Trp Phe Gln Asn Lys Arg Cys
Lys Asp Lys Lys1 5 10 158039PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 80Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Pro Pro Gln Gly Arg Lys1 5 10 15Lys Arg Arg Gln Arg Arg
Arg Pro Pro Gln Gly Arg Lys Lys Arg Arg 20 25 30Gln Arg Arg Arg Pro
Pro Gln 358122PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 81Gly Glu Gln Ile Ala Gln Leu Ile Ala
Gly Tyr Ile Asp Ile Ile Leu1 5 10 15Lys Lys Lys Lys Ser Lys
208236PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 82Lys Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys
Lys Asp Gly Lys1 5 10 15Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser
Val Tyr Val Tyr Lys 20 25 30Val Leu Lys Gln 358336PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 83Lys
Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys1 5 10
15Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys
20 25 30Val Leu Lys Gln 358415PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 84Arg Lys Glu Ser Tyr Ser Val
Tyr Val Tyr Lys Val Leu Lys Gln1 5 10 158533PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 85Lys
Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys1 5 10
15Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys
20 25 30Gln8630PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 86Val Thr Lys Ala Gln Lys Lys Asp Gly
Lys Lys Arg Lys Arg Ser Arg1 5 10 15Lys Glu Ser Tyr Ser Val Tyr Val
Tyr Lys Val Leu Lys Gln 20 25 308727PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 87Ala
Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser1 5 10
15Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln 20
258824PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 88Lys Asp Gly Lys Lys Arg Lys Arg Ser Arg Lys Glu
Ser Tyr Ser Val1 5 10 15Tyr Val Tyr Lys Val Leu Lys Gln
208921PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 89Lys Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser
Val Tyr Val Tyr1 5 10 15Lys Val Leu Lys Gln 209012PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 90Ser
Tyr Ser Val Tyr Val Tyr Lys Val Leu Lys Gln1 5 10919PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 91Val
Tyr Val Tyr Lys Val Leu Lys Gln1 59236PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 92Lys
Gly Ser Lys Lys Ala Val Thr Lys Ala Gln Lys Lys Glu Gly Lys1 5 10
15Lys Arg Lys Arg Ser Arg Lys Glu Ser Tyr Ser Val Tyr Val Tyr Lys
20 25 30Val Leu Lys Gln 35939PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 93Trp Trp His His Lys Lys Arg
Arg Cys1 59418PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 94Trp Trp His His Lys Lys Arg Arg Cys
Cys Arg Arg Lys Lys His His1 5 10 15Trp Trp9513PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 95Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln1 5
10966PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 96Lys Lys Lys Arg Lys Val1 59712PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 97Lys
Lys Lys Arg Lys Val Lys Lys Lys Arg Lys Val1 5 10987PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 98Gly
Arg Lys Lys Arg Arg Cys1 5997PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 99Arg Arg Arg Pro Pro Gln
Cys1 51007PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 100Cys Gly Arg Lys Lys Arg Arg1
51017PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 101Cys Arg Arg Arg Pro Pro Gln1
51026PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 102Trp Lys Lys Lys Lys Cys1 51036PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 103Cys
Trp Lys Lys Lys Lys1 51048PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 104Cys Arg Arg Arg Pro Pro
Gln His1 51057PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 105Cys Arg Arg Arg Pro Pro Gln1
51067PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 106Cys Lys Lys Arg Arg Gln His1
51073PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 107Cys Arg Arg11084PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 108Cys
Arg Arg Arg11095PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 109Cys Arg Arg Arg Arg1
51106PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 110Cys Arg Arg Arg Arg Arg1 51113PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 111Cys
Lys Lys11124PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 112Cys Lys Lys Lys11135PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 113Cys
Lys Lys Lys Lys1 51146PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 114Cys Lys Lys Lys Lys Lys1
51157PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 115Cys Arg Arg Arg Arg Trp Trp1
51166PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 116Cys Arg Arg Arg Trp Trp1 51175PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 117Cys
Arg Arg Trp Trp1 51185PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 118Cys Lys Lys Trp Trp1
51196PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 119Cys Lys Lys Lys Trp Trp1 51207PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 120Cys
Lys Lys Lys Lys Trp Trp1 51218PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 121Cys Trp His His Arg Arg
Lys Lys1 51229PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 122Cys Arg Arg Lys Lys His His Trp Trp1
51236PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 123Cys Lys Lys Arg Arg Trp1 51247PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 124Cys
Lys Lys Arg Arg His Trp1 51258PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 125Cys Lys Lys Arg Arg His
His Trp1 51266PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 126Cys Lys Lys Arg Arg Gln1
51276PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 127Lys Lys Arg Arg Gln Cys1 51287PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 128Cys
Gly Arg Lys Lys Arg Arg1 51297PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 129Gly Arg Lys Lys Arg Arg
Cys1 51308PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 130Cys Gly Arg Lys Lys Arg Arg Gln1
51318PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 131Gln Gly Arg Lys Lys Arg Arg Cys1
51324PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 132Cys Arg Arg His11335PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 133Cys
Arg Arg Arg His1 51346PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 134Cys Arg Arg Arg Arg His1
51357PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 135Cys Arg Arg Arg Arg Arg His1
51364PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 136Cys Lys Lys His11375PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 137Cys
Lys Lys Lys His1 51386PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 138Cys Lys Lys Lys Lys His1
51397PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 139Cys Lys Lys Lys Lys Lys His1
51407PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 140His Trp Lys Lys Arg Arg Cys1
51417PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 141Cys His Trp Lys Lys Arg Arg1
51427PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 142Pro Pro His Arg Arg Arg Cys1
51437PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 143Cys Pro Pro His Arg Arg Arg1
514414PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 144Gly Arg Lys Lys Arg Arg Val Xaa Arg Arg Arg
Pro Pro Gln1 5 1014514PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 145Gly Arg Lys Lys Arg Arg
Val Xaa Arg Arg Lys Lys Arg Gly1 5 1014613PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 146Arg
Arg Arg Pro Pro Gln Val Xaa Pro Pro Arg Arg Arg1 5
1014714PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 147Arg Arg Lys Lys Arg Gly Val Xaa Gly Arg Lys
Lys Arg Arg1 5 1014814PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 148Gln Pro Pro Arg Arg Arg
Val Xaa Arg Arg Arg Pro Pro Gln1 5 1014912PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 149Trp
Lys Lys Lys Lys Val Xaa Lys Lys Lys Lys Trp1 5 1015012PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 150Lys
Lys Lys Lys Trp Val Xaa Trp Lys Lys Lys Lys1 5 1015116PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 151His
Gln Pro Pro Arg Arg Arg Val Xaa Arg Arg Arg Pro Pro Gln His1 5 10
1515214PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 152Gln Pro Pro Arg Arg Arg Val Xaa Arg Arg Arg
Pro Pro Gln1 5 1015314PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 153His Gln Arg Arg Lys Lys
Val Xaa Lys Lys Arg Arg Gln His1 5 101546PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 154Arg
Arg Val Xaa Arg Arg1 51558PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 155Arg Arg Arg Val Xaa Arg
Arg Arg1 515610PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 156Arg Arg Arg Arg Val Xaa Arg Arg Arg
Arg1 5 1015712PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 157Arg Arg Arg Arg Arg Val Xaa Arg Arg
Arg Arg Arg1 5 101586PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 158Lys Lys Val Xaa Lys Lys1
51598PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 159Lys Lys Lys Val Xaa Lys Lys Lys1
516010PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 160Lys Lys Lys Lys Val Xaa Lys Lys Lys Lys1 5
1016112PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 161Lys Lys Lys Lys Lys Val Xaa Lys Lys Lys Lys
Lys1 5 1016214PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 162Trp Trp Arg Arg Arg Arg Val Xaa Arg
Arg Arg Arg Trp Trp1 5 1016312PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 163Trp Trp Arg Arg Arg Val
Xaa Arg Arg Arg Trp Trp1 5 1016410PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 164Trp Trp Arg Arg Val Xaa
Arg Arg Trp Trp1 5 1016510PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 165Trp Trp Lys Lys Val Xaa
Lys Lys Trp Trp1 5 1016612PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 166Trp Trp Lys Lys Lys Val
Xaa Lys Lys Lys Trp Trp1 5 1016714PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 167Trp Trp Lys Lys Lys Lys
Val Xaa Lys Lys Lys Lys Trp Trp1 5 1016816PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 168Lys
Lys Arg Arg His His Trp Val Xaa Trp His His Arg Arg Lys Lys1 5 10
1516918PRTArtificial sequenceDescription of artificial sequence
Synthetic
peptide 169Trp Trp His His Lys Lys Arg Arg Val Xaa Arg Arg Lys Lys
His His1 5 10 15Trp Trp17012PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 170Trp Arg Arg Lys Lys Val
Xaa Lys Lys Arg Arg Trp1 5 1017114PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 171Trp His Arg Arg Lys Lys
Val Xaa Lys Lys Arg Arg His Trp1 5 1017216PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 172Trp
His His Arg Arg Lys Lys Val Xaa Lys Lys Arg Arg His His Trp1 5 10
1517312PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 173Gln Arg Arg Lys Lys Val Xaa Lys Lys Arg Arg
Gln1 5 1017412PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 174Lys Lys Arg Arg Gln Val Xaa Gln Arg
Arg Lys Lys1 5 1017514PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 175Arg Arg Lys Lys Arg Gly
Val Xaa Gly Arg Lys Lys Arg Arg1 5 1017614PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 176Gly
Arg Lys Lys Arg Arg Val Xaa Arg Arg Lys Lys Arg Gly1 5
1017716PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 177Gln Arg Arg Lys Lys Arg Gly Val Xaa Gly Arg
Lys Lys Arg Arg Gln1 5 10 1517816PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 178Gln Gly Arg Lys Lys Arg
Arg Val Xaa Arg Arg Lys Lys Arg Gly Gln1 5 10 151798PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 179His
Arg Arg Val Xaa Arg Arg His1 518010PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 180His
Arg Arg Arg Val Xaa Arg Arg Arg His1 5 1018112PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 181His
Arg Arg Arg Arg Val Xaa Arg Arg Arg Arg His1 5 1018214PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 182His
Arg Arg Arg Arg Arg Val Xaa Arg Arg Arg Arg Arg His1 5
101838PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 183His Lys Lys Val Xaa Lys Lys His1
518410PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 184His Lys Lys Lys Val Xaa Lys Lys Lys His1 5
1018512PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 185His Lys Lys Lys Lys Val Xaa Lys Lys Lys Lys
His1 5 1018614PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 186His Lys Lys Lys Lys Lys Val Xaa Lys
Lys Lys Lys Lys His1 5 1018714PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 187His Trp Lys Lys Arg Arg
Val Xaa Arg Arg Lys Lys Trp His1 5 1018814PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 188Arg
Arg Lys Lys Trp His Val Xaa His Trp Lys Lys Arg Arg1 5
1018914PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 189Pro Pro His Arg Arg Arg Val Xaa Arg Arg Arg
His Pro Pro1 5 1019014PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 190Arg Arg Arg His Pro Pro
Val Xaa Pro Pro His Arg Arg Arg1 5 1019118PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 191Trp
Trp His His Lys Lys Arg Arg Gly Gly Arg Arg Lys Lys His His1 5 10
15Trp Trp1928PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 192Trp Trp His His Lys Lys Arg Arg1
51938PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 193Tyr Tyr His His Lys Lys Arg Arg1
51948PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 194Arg Arg Lys Lys His His Tyr Tyr1
519518PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 195Tyr Tyr His His Lys Lys Arg Arg Cys Cys Arg
Arg Lys Lys His His1 5 10 15Tyr Tyr19617PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 196Tyr
Tyr His His Lys Lys Arg Arg Val Arg Arg Lys Lys His His Tyr1 5 10
15Tyr1974PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 197Trp Trp Arg Arg11984PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 198Arg
Arg Trp Trp11997PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 199Cys Trp Arg Arg Arg Trp Cys1
52009PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 200Cys Trp Trp Arg Arg Arg Trp Trp Cys1
52018PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 201Cys Trp Arg Arg Arg Arg Trp Cys1
520210PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 202Cys Trp Arg Arg His His Arg Arg Trp Cys1 5
1020310PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 203Cys Trp Trp Arg Arg Arg Arg Trp Trp Cys1 5
1020412PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 204Cys Trp Trp Arg Arg His His Arg Arg Trp Trp
Cys1 5 102059PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 205Cys Trp Arg Arg Arg Arg Arg Trp Cys1
520610PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 206Val Gln Ala Ala Ile Asp Tyr Ile Asn Gly1 5
1020711PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 207Cys Trp Trp Arg Arg Arg Arg Arg Trp Trp Cys1 5
1020810PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 208Trp Trp Arg Arg Cys Cys Arg Arg Trp Trp1 5
102099PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 209Trp Trp Arg Arg Val Arg Arg Trp Trp1
52104PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 210Tyr Tyr Arg Arg12114PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 211Arg
Arg Tyr Tyr12127PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 212Cys Tyr Arg Arg Arg Tyr Cys1
52139PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 213Cys Tyr Tyr Arg Arg Arg Tyr Tyr Cys1
52148PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 214Cys Tyr Arg Arg Arg Arg Tyr Cys1
521510PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 215Cys Tyr Arg Arg His His Arg Arg Tyr Cys1 5
1021610PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 216Cys Tyr Tyr Arg Arg Arg Arg Tyr Tyr Cys1 5
1021712PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 217Cys Tyr Tyr Arg Arg His His Arg Arg Tyr Tyr
Cys1 5 102189PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 218Cys Tyr Arg Arg Arg Arg Arg Tyr Cys1
521911PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 219Cys Tyr Tyr Arg Arg Arg Arg Arg Tyr Tyr Cys1 5
1022010PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 220Cys Tyr Tyr Arg Arg Arg Arg Tyr Tyr Cys1 5
1022110PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 221Tyr Tyr Arg Arg Cys Cys Arg Arg Tyr Tyr1 5
102229PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 222Tyr Tyr Arg Arg Val Arg Arg Tyr Tyr1
52236PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 223Trp Trp Arg Arg His His1 52246PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 224His
His Arg Arg Trp Trp1 522514PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 225Trp Trp Arg Arg His His
Cys Cys His His Arg Arg Trp Trp1 5 1022613PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 226Trp
Trp Arg Arg His His Val His His Arg Arg Trp Trp1 5
102276PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 227Tyr Tyr Arg Arg His His1 52286PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 228His
His Arg Arg Tyr Tyr1 522914PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 229Tyr Tyr Arg Arg His His
Cys Cys Arg Arg His His Tyr Tyr1 5 1023013PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 230Tyr
Tyr Arg Arg His His Val Arg Arg His His Tyr Tyr1 5
102315PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 231Trp Trp Arg Arg Arg1 52325PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 232Arg
Arg Arg Trp Trp1 523312PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 233Trp Trp Arg Arg Arg Cys
Cys Arg Arg Arg Trp Trp1 5 1023411PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 234Trp Trp Arg Arg Arg Val
Arg Arg Arg Trp Trp1 5 102355PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 235Tyr Tyr Arg Arg Arg1
52365PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 236Arg Arg Arg Tyr Tyr1 523712PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 237Tyr
Tyr Arg Arg Arg Cys Cys Arg Arg Arg Tyr Tyr1 5 1023811PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 238Tyr
Tyr Arg Arg Arg Val Arg Arg Arg Tyr Tyr1 5 102397PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 239Trp
Trp Arg Arg Arg His His1 52407PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 240His His Arg Arg Arg Trp
Trp1 524116PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 241Trp Trp Arg Arg Arg His His Cys Cys His His
Arg Arg Arg Trp Trp1 5 10 1524215PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 242Trp Trp Arg Arg Arg His
His Val His His Arg Arg Arg Trp Trp1 5 10 152437PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 243Tyr
Tyr Arg Arg Arg His His1 52447PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 244His His Arg Arg Arg Tyr
Tyr1 524516PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 245Tyr Tyr Arg Arg Arg His His Cys Cys Arg Arg
Arg His His Tyr Tyr1 5 10 1524615PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 246Tyr Tyr Arg Arg Arg His
His Val Arg Arg Arg His His Tyr Tyr1 5 10 152476PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 247Trp
Trp Arg Arg Arg Arg1 52486PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 248Arg Arg Arg Arg Trp Trp1
524914PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 249Trp Trp Arg Arg Arg Arg Cys Cys Arg Arg Arg
Arg Trp Trp1 5 1025013PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 250Trp Trp Arg Arg Arg Arg
Val Arg Arg Arg Arg Trp Trp1 5 102516PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 251Tyr
Tyr Arg Arg Arg Arg1 52526PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 252Arg Arg Arg Arg Tyr Tyr1
525314PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 253Tyr Tyr Arg Arg Arg Arg Cys Cys Arg Arg Arg
Arg Tyr Tyr1 5 1025413PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 254Tyr Tyr Arg Arg Arg Arg
Val Arg Arg Arg Arg Tyr Tyr1 5 102558PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 255Trp
Trp Arg Arg Arg Arg His His1 52568PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 256His His Arg Arg Arg Arg
Trp Trp1 525718PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 257Trp Trp Arg Arg Arg Arg His His Cys
Cys His His Arg Arg Arg Arg1 5 10 15Trp Trp25817PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 258Trp
Trp Arg Arg Arg Arg His His Val His His Arg Arg Arg Arg Trp1 5 10
15Trp2598PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 259Tyr Tyr Arg Arg Arg Arg His His1
52608PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 260His His Arg Arg Arg Arg Tyr Tyr1
526118PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 261Tyr Tyr Arg Arg Arg Arg His His Cys Cys Arg
Arg Arg Arg His His1 5 10 15Tyr Tyr26217PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 262Tyr
Tyr Arg Arg Arg Arg His His Val Arg Arg Arg Arg His His Tyr1 5 10
15Tyr26319PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 263Trp Trp His His Lys Lys Arg Arg Trp Val Trp
Arg Arg Lys Lys His1 5 10 15His Trp Trp2647PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 264Trp
Trp His His Arg Arg Cys1 526513PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 265Trp Trp His His Arg Arg
Val Arg Arg His His Trp Trp1 5 102669PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 266Trp
Trp His His His Arg Arg Arg Cys1 526710PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 267Cys
Trp Trp His His His Arg Arg Arg Cys1 5 1026811PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 268Cys
Trp Trp Trp His His His His Arg Arg Arg1 5 102699PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 269Cys
Trp Trp Trp Lys Lys Arg Arg Arg1 52708PRTArtificial
sequenceDescription of artificial sequence Synthetic peptide 270Cys
Lys Lys Lys Trp Arg Arg Trp1 52718PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 271Cys Trp Arg Arg Arg Trp
Arg Arg1 527210PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 272Cys Trp Trp His His Lys Lys Arg Arg
Cys1 5 1027310PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 273Trp Trp Cys His His Lys Lys Cys Arg
Arg1 5 1027410PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 274Trp Trp His His Cys Lys Lys Arg Arg
Cys1 5 1027510PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 275Cys Trp Trp His His Lys Lys Cys Arg
Arg1 5 1027610PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 276Trp Trp His His Cys Cys Lys Lys Arg
Arg1 5 102779PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 277Arg Arg Trp Trp Lys Lys His His Cys1
527810PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 278Cys Trp Trp His His Lys Lys Lys Lys Cys1 5
1027910PRTArtificial sequenceDescription of artificial sequence
Synthetic
peptide 279Cys Trp Trp His His Arg Arg Arg Arg Cys1 5
102808PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 280Cys Arg Arg Arg Arg His His Cys1
52818PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 281Cys His His Lys Lys Lys Lys Cys1
52828PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 282Cys His His Arg Arg Arg Arg Cys1
528310PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 283Cys Tyr Tyr Arg Arg Arg Arg His His Cys1 5
1028410PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 284Cys Tyr Tyr Lys Lys Lys Lys His His Cys1 5
1028523PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 285Cys Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Cys
2028624PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 286Cys His Trp Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Lys Cys
2028716PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 287Ala Ala Ala Asx Xaa Tyr Xaa Gln Trp Leu Xaa
Xaa Xaa Gly Pro Xaa1 5 10 1528822PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 288Cys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys
Cys 2028924PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 289Cys His Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys His Cys
2029027PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 290Gly Ala Leu Phe Leu Ala Phe Leu Ala Ala Ala
Leu Ser Leu Met Gly1 5 10 15Leu Trp Ser Gln Pro Lys Lys Lys Arg Lys
Val 20 2529127PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 291Gly Ala Leu Phe Leu Ala Phe Leu Ala
Ala Ala Leu Xaa Leu Met Gly1 5 10 15Leu Trp Xaa Gln Xaa Lys Lys Lys
Arg Lys Val 20 2529210PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 292Trp Ser Gln Pro Lys Lys
Lys Arg Lys Val1 5 1029312PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 293Xaa Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val Xaa1 5 1029410PRTArtificial sequenceDescription
of artificial sequence Synthetic peptide 294Trp Xaa Gln Pro Lys Lys
Lys Arg Lys Xaa1 5 1029510PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 295Xaa Xaa Gln Pro Lys Lys
Lys Arg Lys Val1 5 1029623PRTArtificial sequenceDescription of
artificial sequence Synthetic peptide 296Leu Ile Arg Leu Trp Ser
His Leu Ile His Ile Trp Phe Gln Asn Arg1 5 10 15Arg Leu Lys Trp Lys
Lys Lys 2029723PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 297Leu Ile Arg Leu Trp Xaa His Leu Ile
His Ile Trp Phe Gln Xaa Arg1 5 10 15Arg Leu Lys Trp Lys Lys Lys
2029810PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 298Gln Asn Arg Arg Leu Lys Trp Lys Lys Lys1 5
1029912PRTArtificial sequenceDescription of artificial sequence
Synthetic peptide 299Xaa Gln Asn Arg Arg Leu Lys Trp Lys Lys Lys
Xaa1 5 1030010PRTArtificial sequenceDescription of artificial
sequence Synthetic peptide 300Gln Xaa Arg Arg Leu Lys Trp Lys Lys
Lys1 5 1030121DNAArtificial sequenceDescription of combined DNA/RNA
molecule Synthetic primer 301ccgucagccg auuugcuaut t
2130221DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic primer 302auagcaaauc ggcugacggt t
2130321DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic primer 303gggucggaac ccaagcuuat t
2130421DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic primer 304uaagcuuggg uuccgaccct a
2130521DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 305cuacacaaau cagcgauuut t
2130621DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 306aaaucgcuga uuuguguagt c
2130721DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 307cgggacucua gcauacuuat t
2130821DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 308uaaguaugcu agagucccgt t
2130921DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 309acugacagcc agacagcgat t
2131021DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 310ucgcugucug gcugucagut t
2131121DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 311agacagcgac caaaagaaut t
2131221DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 312auucuuuugg ucgcugucut t
2131321DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 313augaagaucu guuccaccat t
2131421DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 314ugguggaaca gaucuucaut t
2131521DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 315gaucuguucc accauugaat t
2131621DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 316uucaauggug gaacagauct t
2131721DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 317gcaauugagg agugccugat t
2131821DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 318ucaggcacuc cucaauugct t
2131921DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 319uugaggagug ccugauuaat t
2132021DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 320uuaaucaggc acuccucaat t
2132121DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 321ggaucuuauu ucuucggagt t
2132221DNAArtificial SequenceDescription of combined DNA/RNA
molecule Synthetic oligonucleotide 322cuccgaagaa auaagaucct t
2132320DNAArtificial SequenceDescription of artificial sequence
Synthetic primer 323ggctcccagt tcttcatcac 2032416DNAArtificial
SequenceDescription of artificial sequence Synthetic primer
324ccttccgcac cacctc 1632526DNAArtificial SequenceDescription of
artificial sequence Synthetic probe 325ctagatggca agcatgtggt gtttgg
2632624DNAArtificial SequenceDescription of artificial sequence
Synthetic primer 326tctatcatca acgggtacaa acga 2432722DNAArtificial
SequenceDescription of artificial sequence Synthetic primer
327cttttcagca agtgggaagg tg 2232827DNAArtificial
SequenceDescription of artificial sequence Synthetic probe
328cctggccttg tctgtggaga cggatta 2732925DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 329ggaucuuauu ucuucggaga caatg 2533027RNAArtificial
SequenceDescription of artificial sequence Synthetic
oligonucleotide 330cauugucucc gaagaaauaa gauccuu
2733121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 331uucuccgaac gugucacgut t
2133221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 332acgugacacg uucggagaat t
2133321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 333cuacacaaau cagcgauuut t
2133421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 334aaaucgcuga uuuguguagt c
2133521RNAArtificial SequenceDescription of artificial sequence
Synthetic oligonucleotide 335ggaaagacug uuccaaaaau u
2133621RNAArtificial SequenceDescription of artificial sequence
Synthetic oligonucleotide 336uuuuuggaac agucuuuccu u
2133721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 337ggaucuuauu ucuucggagt t
2133821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 338cuccgaagaa auaagaucct t
2133921DNAArtificial SequenceDescription of artificial sequence
Synthetic oligonucleotide 339ccgtcagccg atttgctatt t
2134021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 340auagcaaatc ggctgacggt t
21341100PRTArtificial SequenceDescription of artificial sequence
Synthetic polypeptide 341Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg 20 25 30Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 50 55 60Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg65 70 75 80Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 85 90 95Arg Arg Arg
Arg 100342100PRTArtificial SequenceDescription of artificial
sequence Synthetic polypeptide 342Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 30Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 50 55 60Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys65 70 75 80Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 85 90 95Lys
Lys Lys Lys 100343100PRTArtificial SequenceDescription of
artificial sequence Synthetic polypeptide 343His His His His His
His His His His His His His His His His His1 5 10 15His His His His
His His His His His His His His His His His His 20 25 30His His His
His His His His His His His His His His His His His 35 40 45His His
His His His His His His His His His His His His His His 50 55 60His
His His His His His His His His His His His His His His His65 70 75
80His His His His His His His His His His His His His His His His
85 90 95His His His His 100344102PRTArtificial SequenceDescription
of artificial sequence Synthetic polypeptide 344His Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg1 5 10 15Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 50 55 60Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg65 70 75
80Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
85 90 95Arg Arg Arg Arg Arg His 100345102PRTArtificial
SequenceDescription of artificial sequence Synthetic polypeptide
345His Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1
5 10 15Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys 20 25 30Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys 35 40 45Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys 50 55 60Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys65 70 75 80Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys 85 90 95Lys Lys Lys Lys Lys His
100346300PRTArtificial SequenceDescription of artificial sequence
Synthetic polypeptide 346His His His His His His His His His His
His His His His His His1 5 10 15His His His His His His His His His
His His His His His His His 20 25 30His His His His His His His His
His His His His His His His His 35 40 45His His His His His His His
His His His His His His His His His 50 55 60His His His His His His
His His His His His His His His His His65 70 75 80His His His His
His His His His His His His His His His His His 85 90 95His His His
His Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 100 105 110Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 115 120
125Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
130 135 140Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg145 150 155 160Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg 165 170 175Arg Arg Arg Arg Arg Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg 180 185 190Arg Arg Arg Arg Arg Arg Arg
Arg His His His His His His His His 195 200 205His His His His His
His His His His His His His His His His His 210 215 220His His His
His His His His His His His His His His His His His225 230 235
240His His His His His His His His His His His His His His His His
245 250 255His His His His His His His His His His His His His His
His His 260 265 270His His His His His His His His His His His His
His His His His 275 280 285His His His His His His His His His His
His His 290 295 300347300PRTArtificial SequenceDescription of
artificial sequence Synthetic polypeptide 347His His His His His
His His His His His His His His His His His1 5 10 15His His His His
His His His His His His His His His His His His 20 25 30His
His His His His His His His His His His His His His His His 35 40
45His His His His His His His His His His His His His His His His
50 55 60His His His His His His His His His His His His His His His
His65 70 75 80His His His His His His His His His His His His His
His His His 85 90 95His His His His Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys 100 105 110Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys 115 120 125Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys 130 135 140Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys145 150 155 160Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 165 170 175Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 180 185
190Lys Lys Lys Lys Lys Lys Lys Lys His His His His His His His His
195 200 205His His His His His His His His His His His His His His
His His 210 215 220His His His His His His His His His His His His
His His His His225 230 235 240His His His His His His His His His
His His His His His His His 245 250 255His His His His His His His
His His His His His His His His His 260 265 270His His His His His
His His His His His His His His His His His 275 280 285His His His
His His His His His His His His His 290 295 30034840PRTArtificial
SequenceDescription of artificial sequence Synthetic peptide 348His
Arg His Arg His Arg His Arg His Arg His Arg His Arg His Arg1 5 10
15His Arg His Arg His Arg His Arg His Arg His Arg His Arg His Arg
20 25 30His Arg His Arg His Arg His Arg 35 4034940PRTArtificial
SequenceDescription of artificial sequence Synthetic peptide 349His
Lys His Lys His Lys His Lys His Lys His Lys His Lys His Lys1 5 10
15His Lys His Lys His Lys His Lys His Lys His Lys His Lys His Lys
20 25 30His Lys His Lys His Lys His Lys 35 4035013PRTArtificial
SequenceDescription of artificial sequence Synthetic peptide 350His
His His His Arg Arg Arg Arg Arg Arg Arg Arg Xaa1 5
1035116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 351His His His His His His His His Arg Arg Arg
Arg Arg Arg Arg Arg1 5 10 1535228PRTArtificial SequenceDescription
of artificial sequence Synthetic peptide 352Gly Ala Leu Phe Leu Ala
Phe Leu Ala Ala Ala Leu Ser Leu Met Gly1 5 10 15Leu Trp Ser Gln Pro
Lys Lys Lys Arg Lys Val Xaa 20 2535328PRTArtificial
SequenceDescription of artificial sequence Synthetic peptide 353Gly
Ala Leu Phe Leu Ala Phe Leu Ala Ala Ala Leu Ser Leu Met Gly1 5 10
15Leu Trp Ser Gln Pro Lys Ser Lys Arg Lys Val Xaa 20
2535419PRTArtificial SequenceDescription of artificial sequence
Synthetic peptide 354His His His His His Lys His His His Lys Lys
Lys His Lys His Lys1 5 10 15Lys Lys Xaa35519PRTArtificial
SequenceDescription of artificial sequence Synthetic peptide 355His
His His His His Lys His His His Lys Lys Lys His Lys His Lys1 5 10
15Lys Lys Xaa35627PRTArtificial SequenceDescription of artificial
sequence Synthetic peptide 356Gly Ala Leu Phe Leu Gly Phe Leu Gly
Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala Trp Ser Gln Pro Lys Ser Lys
Arg Lys Val 20 25
* * * * *