U.S. patent application number 11/121566 was filed with the patent office on 2006-02-23 for compostions and methods for enhancing delivery of nucleic acids into cells and for modifying expression of target genes in cells.
Invention is credited to Lishan Chen, Yuching Chen, Kunyuan Cui, Sasha J. Mayer.
Application Number | 20060040882 11/121566 |
Document ID | / |
Family ID | 36119611 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040882 |
Kind Code |
A1 |
Chen; Lishan ; et
al. |
February 23, 2006 |
Compostions and methods for enhancing delivery of nucleic acids
into cells and for modifying expression of target genes in
cells
Abstract
Polynucleotide delivery-enhancing polypeptides are admixed or
complexed with, or conjugated to, nucleic acids for enhancing
delivery the nucleic acids into cells. The transported nucleic
acids are active in target cells as small inhibitory nucleic acids
(siNAs) that modulate expression of target genes, mediated at least
in part by RNA interference (RNAi). The siNA/polypeptide
compositions and methods of the invention provide effective tools
to modulate gene expression and alter phenotype in mammalian cells,
including by altering phenotype in a manner that eliminates disease
symptoms or alters disease potential in targeted cells or subject
individuals to which the siNA/polypeptide compositions are
administered.
Inventors: |
Chen; Lishan; (Bellevue,
WA) ; Cui; Kunyuan; (Bothell, WA) ; Chen;
Yuching; (Bellevue, WA) ; Mayer; Sasha J.;
(Snohomish, WA) |
Correspondence
Address: |
Peter J. Knudsen, Esq.;Patent Counsel
Nastech Pharmaceutical Company Inc.
3450 Monte Villa Parkway
Bothell
WA
98021-8909
US
|
Family ID: |
36119611 |
Appl. No.: |
11/121566 |
Filed: |
May 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60568027 |
May 4, 2004 |
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60570512 |
May 12, 2004 |
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60570513 |
May 12, 2004 |
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60613416 |
Sep 27, 2004 |
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60656572 |
Feb 25, 2005 |
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60667833 |
Apr 1, 2005 |
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Current U.S.
Class: |
514/44A ;
435/455 |
Current CPC
Class: |
C12N 15/1136 20130101;
A61P 37/00 20180101; C12N 15/111 20130101; C07K 14/43572 20130101;
A61P 19/02 20180101; A61K 2121/00 20130101; A61P 17/06 20180101;
C07K 14/47 20130101; C12N 2310/14 20130101; C12N 2320/32 20130101;
C07K 14/001 20130101; C12N 2310/3513 20130101; A61K 47/64 20170801;
A61P 43/00 20180101; A61P 29/00 20180101; C12N 15/87 20130101 |
Class at
Publication: |
514/044 ;
435/455 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/85 20060101 C12N015/85 |
Claims
1. A method for causing uptake of a double stranded nucleic acid
into an animal cell, which comprises incubating said cells with a
mixture comprising a polynucleotide delivery-enhancing polypeptide
and said nucleic acid.
2. The method of claim 1, wherein the nucleic acid is admixed,
complexed or conjugated with the polynucleotide delivery-enhancing
polypeptide.
3. The method of claim 1, wherein said nucleic acid is a small
inhibitory RNA (siRNA).
4. The method of claim 3, wherein the nucleic acid comprises a
siRNA that is complementary to a portion of a TNF-gene.
5. The method of claim 1, wherein the nucleic acid has a length of
30 or fewer nucleotides or nucleotide base pairs.
6. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises a histone protein, or a
polypeptide or peptide fragment, derivative, analog, or conjugate
thereof.
7. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises an amphipathic amino acid
sequence.
8. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises a protein transduction
domain or motif
9. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises a fusogenic peptide domain
or motif.
10. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises a DNA-binding domain or
motif.
11. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more amino acid
sequences listed in Tables 2-8 above.
12. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more histone
proteins selected from histone H1, histone H2B, histone H3, and
histone H41, or a fragment thereof, an amino acid sequence selected
from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO:
29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30),
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and
WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4:1,
MW 20,000-50,000), Poly Orn-Trp (4:1, MW 20,000-50,000), or
mellitin.
13. The method of claim 1, wherein the polynucleotide
delivery-enhancing polypeptide is pegylated.
14. The method of claim 1, wherein said mixture further comprises a
cationic lipid.
15. The method of claim 14, wherein the cationic lipid is
Lipofectin.RTM. or Lipofectamine.RTM..
16. A composition comprising a polynucleotide delivery-enhancing
polypeptide and a double stranded nucleic acid, wherein said
composition causes uptake of said nucleic acid into an animal
cell.
17. The composition of claim 16, wherein the nucleic acid is
admixed, complexed or conjugated with the polynucleotide
delivery-enhancing polypeptide.
18. The composition of claim 16, wherein said nucleic acid is a
small inhibitory RNA (siRNA).
19. The composition of claim 18, wherein the nucleic acid comprises
a siRNA that is complementary to a portion of a TNF-gene.
20. The composition of claim 16, wherein the nucleic acid has a
length of 30 or fewer nucleotides or nucleotide base pairs.
21. The composition of claim 16, wherein the polynucleotide
delivery-enhancing polypeptide comprises a histone protein, or a
polypeptide or peptide fragment, derivative, analog, or conjugate
thereof.
22. The composition of claim 1 6, wherein the polynucleotide
delivery-enhancing polypeptide comprises an amphipathic amino acid
sequence.
23. The composition of claim 16, wherein the polynucleotide
delivery-enhancing polypeptide comprises a protein transduction
domain or motif
24. The composition of claim 16, wherein the polynucleotide
delivery-enhancing polypeptide comprises a fusogenic peptide domain
or motif.
25. The composition of claim 1 6, wherein the polynucleotide
delivery-enhancing polypeptide comprises a DNA-binding domain or
motif.
26. The composition of claim 16, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more amino acid
sequences listed in Tables 2-8 above.
27. The composition of claim 1 6, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more histone
proteins selected from histone H1, histone H2B, histone H3, and
histone H41, or a fragment thereof, an amino acid sequence selected
from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO:
29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30),
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and
WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4:1,
MW 20,000-50,000), Poly Orn-Trp (4:1, MW 20,000-50,000), or
mellitin.
28. The composition of claim 1 6, wherein the polynucleotide
delivery-enhancing polypeptide is pegylated.
29. The composition of claim 1 6, further comprising a cationic
lipid.
30. The composition of claim 29, wherein the cationic lipid is
Lipofectin.RTM. or Lipofectamine.RTM..
31. A method for modifying expression of a target gene in an animal
cell, which comprises incubating said cell with a mixture
comprising a polynucleotide delivery-enhancing polypeptide and a
nucleic acid, wherein said nucleic acid is complementary to a
region of said target gene.
32. The method of claim 31, wherein the nucleic acid is admixed,
complexed or conjugated with the polynucleotide delivery-enhancing
polypeptide.
33. The method of claim 31, wherein said nucleic acid is a small
inhibitory RNA (siRNA).
34. The method of claim 33, wherein the nucleic acid comprises a
siRNA that is complementary to a portion of a TNF-gene.
35. The method of claim 31, wherein the nucleic acid has a length
of 30 or fewer nucleotides or nucleotide base pairs.
36. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises a histone protein, or a
polypeptide or peptide fragment, derivative, analog, or conjugate
thereof.
37. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises an amphipathic amino acid
sequence.
38. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises a protein transduction
domain or motif
39. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises a fusogenic peptide domain
or motif.
40. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises a DNA-binding domain or
motif.
41. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more amino acid
sequences listed in Tables 2-8 above.
42. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more histone
proteins selected from histone H1, histone H2B, histone H3, and
histone H41, or a fragment thereof, an amino acid sequence selected
from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO:
29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30),
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and
WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4:1,
MW 20,000-50,000), Poly Orn-Trp (4:1, MW 20,000-50,000), or
mellitin.
43. The method of claim 31, wherein the polynucleotide
delivery-enhancing polypeptide is pegylated.
44. The method of claim 31, wherein said mixture further comprises
a cationic lipid.
45. The method of claim 44, wherein the cationic lipid is
Lipofectin.RTM. or Lipofectamine.RTM..
46. A composition comprising a polynucleotide delivery-enhancing
polypeptide and a double stranded nucleic acid, wherein said
composition causes uptake of said nucleic acid into an animal cell,
wherein said nucleic acid is complementary to a region of a target
gene and modifies expression of said target gene in said cell.
47. A method for changing a phenotype of an animal subject, which
comprises administering to said subject a mixture of a
polynucleotide delivery-enhancing polypeptide and a double stranded
nucleic acid, wherein said nucleic acid is complementary to a
region of a target gene in said subject.
48. The method of claim 47, wherein said subject is an animal cell
or individual.
49. The method of claim 47, wherein the nucleic acid is admixed,
complexed or conjugated with the polynucleotide delivery-enhancing
polypeptide.
50. The method of claim 47, wherein said nucleic acid is a small
inhibitory RNA (siRNA).
51. The method of claim 50, wherein the nucleic acid comprises a
siRNA that is complementary to a portion of a TNF-gene.
52. The method of claim 47, wherein the nucleic acid has a length
of 30 or fewer nucleotides or nucleotide base pairs.
53. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises a histone protein, or a
polypeptide or peptide fragment, derivative, analog, or conjugate
thereof.
54. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises an amphipathic amino acid
sequence.
55. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises a protein transduction
domain or motif
56. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises a fusogenic peptide domain
or motif.
57. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises a DNA-binding domain or
motif.
58. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more amino acid
sequences listed in Tables 2-8 above.
59. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more histone
proteins selected from histone H1, histone H2B, histone H3, and
histone H41, or a fragment thereof, an amino acid sequence selected
from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO:
29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30),
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and
WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4:1,
MW 20,000-50,000), Poly Orn-Trp (4:1, MW 20,000-50,000), or
mellitin.
60. The method of claim 47, wherein the polynucleotide
delivery-enhancing polypeptide is pegylated.
61. The method of claim 47, wherein said mixture further comprises
a cationic lipid.
62. The method of claim 61, wherein the cationic lipid is
Lipofectin.RTM. or Lipofectamine.RTM..
63. A mixture comprising a polynucleotide delivery-enhancing
polypeptide and a double stranded nucleic acid, wherein said
mixture causes uptake of said nucleic acid into cells of an animal,
wherein said nucleic acid is complementary to a region of a target
gene in said cell and is active to modulate expression of said
target gene to mediate a change in phenotype of the cell or
animal.
64. A method for treating a disease or adverse condition in an
animal subject comprising administering to said subject an
effective amount of a mixture comprising a polynucleotide
delivery-enhancing polypeptide and a double stranded nucleic acid,
wherein said mixture causes uptake of said nucleic acid into cells
of the subject, and wherein said nucleic acid is complementary to a
region of a target gene in said cells and is active to modulate
expression of said target gene to mediate prevention or reduction
in the occurrence or severity of one or more symptoms of said
disease or condition in the subject.
65. The method of claim 64, wherein said subject is an animal cell
or individual.
66. The method of claim 64, wherein the nucleic acid is admixed,
complexed or conjugated with the polynucleotide delivery-enhancing
polypeptide.
67. The method of claim 64, wherein said nucleic acid is a small
inhibitory RNA (siRNA).
68. The method of claim 64, wherein the nucleic acid comprises a
siRNA that is complementary to a portion of a TNF-gene.
69. The method of claim 64, wherein the nucleic acid has a length
of 30 or fewer nucleotides or nucleotide base pairs.
70. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises a histone protein, or a
polypeptide or peptide fragment, derivative, analog, or conjugate
thereof.
71. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises an amphipathic amino acid
sequence.
72. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises a protein transduction
domain or motif
73. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises a fusogenic peptide domain
or motif.
74. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises a DNA-binding domain or
motif.
75. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more amino acid
sequences listed in Tables 2-8 above.
76. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide comprises one or more histone
proteins selected from histone H1, histone H2B, histone H3, and
histone H41, or a fragment thereof, an amino acid sequence selected
from GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO:
29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30),
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), and
WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO: 27), Poly Lys-Trp (4:1,
MW 20,000-50,000), Poly Orn-Trp (4:1, MW 20,000-50,000), or
mellitin.
77. The method of claim 64, wherein the polynucleotide
delivery-enhancing polypeptide is pegylated.
78. The method of claim 64, wherein said mixture further comprises
a cationic lipid.
79. The method of claim 78, wherein the cationic lipid is
Lipofectin.RTM. or Lipofectamine.RTM..
80. A composition comprising a double-stranded nucleic acid (dsNA)
having a sense and an antisense strand complexed or covalently
bonded to one or more polynucleotide delivery-enhancing
polypeptides.
81. The composition of claim 80, wherein the dsNA anti-sense strand
can hybridize to an mRNA present within a cell of interest.
82. The composition of claim 80, wherein each strand of the dsNA
has 30 or fewer nucleotide pairs.
83. The composition of claim 80, wherein each NA complex of claim 2
wherein each strand of the dsNA has a length of between about 19-25
nucleotides.
84. The composition of claim 80, wherein the dsNA comprises a small
inhibitory RNA (siRNA).
85. The composition of claim 80, wherein the dsNA is a
double-stranded (ds) hybrid nucleic acid (ds Hybrid) or has a sense
and an antisense strand wherein one of the strands is a strand of
DNA and the other strand is a strand of RNA.
86. A composition comprising a double-stranded nucleic acid (dsNA)
having a sense and an antisense strand admixed, complexed, or
covalently bonded with one or more polynucleotide
delivery-enhancing polypeptides and a cationic lipid.
87. The composition of claim 86, wherein the cationic lipid is
selected from the group consisting of
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride,
1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane,
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide,
dimethyldioctadecylammonium bromide,
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoracetate,
1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid,
5-carboxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoyl
spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl
spermine, tetramethyltetramyristyl spermine and tetramethyidioleyl
spermine, DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl
ammonium chloride), DOTAP
(1,2-bis(oleoyloxy)-3,3-(trimethylammonium)propane), DMRIE
(1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium
bromide), DDAB (dimethyl dioctadecyl ammonium bromide), polyvalent
cationic lipids, lipospermines, DOSPA
(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,
N-dimethyl-1-propanaminium trifluoro-acetate), DOSPER
(1,3-dioleoyloxy-2-(6carboxy spermyl)-propyl-amid, di- and
tetra-alkyl-tetra-methyl spermines, TMTPS
(tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl
spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS
(tetramethyltetramyristyl spermine), TMDOS (tetramethyldioleyl
spermine) DOGS (dioctadecyl-amidoglycylspermine (TRANSFECTAM.RTM.),
cationic lipids combined with non-cationic lipids, DOPE
(dioleoylphosphatidylethanolamine), DPhPE
(diphytanoylphosphatidylethanolamine) or cholesterol, a cationic
lipid composition composed of a 3:1 (w/w) mixture of DOSPA and
DOPE, and a 1:1 (w/w) mixture of DOTMA and DOPE.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Patent Application No. 60/568,027, filed May 4, 2004,
U.S. Provisional Patent Application No. 60/570,512, filed May 12,
2004, U.S. Provisional Patent Application No. 60/570,513, filed May
12, 2004, U.S. Provisional Patent Application No. 60/613,416, filed
Sep. 27, 2004, U.S. Provisional Patent Application No. 60/656,572
filed Feb. 25, 2005, and U.S. Provisional Patent Application No.
60/667,833, filed Apr. 1, 2005, the disclosures of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The invention relates to methods and compositions for
delivering nucleic acids into cells. More specifically, the
invention relates to procedures and preparations for delivering
double-stranded polynucleotides into cells to modify expression of
target genes to alter a phenotype, such as a disease state or
potential, of the cells.
BACKGROUND OF THE INVENTION
[0003] Delivering nucleic acids into animal and plant cells has
long been an important object of molecular biology research and
development. Recent developments in the areas of gene therapy,
antisense therapy and RNA interference (RNAi) therapy have created
a need to develop more efficient means for introducing nucleic
acids into cells.
[0004] A diverse array of plasmids and other nucleic acid "vectors"
have been developed for delivering large polynucleotide molecules
into cells. Typically these vectors incorporate large DNA molecules
comprising intact genes for the purpose of transforming target
cells to express a gene of scientific or therapeutic interest.
[0005] The process by which exogenous nucleic acids are delivered
artificially into cells is generally referred to as transfection.
Cells can be transfected to uptake a functional nucleic acid from
an exogenous source using a variety of techniques and materials.
The most commonly used transfection methods are calcium phosphate
transfection, and electroporation. A variety of other methods for
tranducing cells to deliver exogenous DNA or RNA molecules have
been developed, including viral-mediated transduction, cationic
lipid or liposomal delivery, and numerous methods that target
mechanical or biochemical membrane disruption/penetration (e.g.,
using detergents, microinjection, or particle guns).
[0006] RNA interference is a process of sequence-specific post
transcriptional gene silencing in cells initiated by a
double-stranded (ds) polynucleotide, usually a dsRNA, that is
homologous in sequence to a portion of a targeted messenger RNA
(mRNA). Introduction of a suitable dsRNA into cells leads to
destruction of endogenous, cognate mRNAs (i.e., mRNAs that share
substantial sequence identity with the introduced dsRNA). The dsRNA
molecules are cleaved by an RNase III family nuclease called dicer
into short-interfering RNAs (siRNAs), which are 19-23 nucleotides
(nt) in length. The siRNAs are then incorporated into a
multicomponent nuclease complex known as the RNA-induced silencing
complex or "RISC". The RISC identifies mRNA substrates through
their homology to the siRNA, and effectuates silencing of gene
expression by binding to and destroying the targeted mRNA.
[0007] RNA interference is emerging a promising technology for
modifying expression of specific genes in plant and animal cells,
and is therefore expected to provide useful tools to treat a wide
range of diseases and disorders amenable to treatment by
modification of endogenous gene expression.
[0008] There remains a long-standing need in the art for better
tools and methods to deliver siRNAs and other small inhibitory
nucleic acids (siNAs) into cells, particularly in view of the fact
that existing techniques for delivering nucleic acids to cells are
limited by poor efficiency and/or high toxicity of the delivery
reagents. Related needs exist for improved methods and formulations
to deliver siNAs in an effective amount, in an active and enduring
state, and using non-toxic delivery vehicles, to selected cells,
tissues, or compartments to mediate regulation of gene expression
in a manner that will alter a phenotype or disease state of the
targeted cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 illustrates peptide-mediated uptake of siNAs
complexed or conjugated to a polynucleotide delivery-enhancing
polypeptide of the invention.
[0010] FIG. 2 further illustrates peptide-mediated uptake of siNAs
complexed or conjugated to a polynucleotide delivery-enhancing
polypeptide of the invention.
[0011] FIG. 3 shows paw data for siRNA/peptide injected mice which
demonstrate delayed RA progression in the treated mice comparable
to that exhibited by Ramicade-treated subjects.
[0012] FIG. 4 provides results of uptake efficacy and viability
studies in mouse fibroblasts for PN73 rationally-designed
derivative polynucleotide delivery-enhancing polypeptides of the
invention.
DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION
[0013] The present invention satisfies these needs and fulfills
additional objects and advantages by providing novel compositions
and methods that employ a short interfering nucleic acid (siNA), or
a precursor thereof, in combination with a polynucleotide
delivery-enhancing polypeptide. The polynucleotide
delivery-enhancing polypeptide is a natural or artificial
polypeptide selected for its ability to enhance intracellular
delivery or uptake of polynucleotides, including siNAs and their
precursors.
[0014] Within the novel compositions of the invention, the siNA may
be admixed or complexed with, or conjugated to, the polynucleotide
delivery-enhancing polypeptide to form a composition that enhances
intracellular delivery of the siNA as compared to delivery
resulting from contacting the target cells with a naked siNA (i.e.,
siNA without the delivery-enhancing polypeptide present).
[0015] In certain embodiments of the invention, the polynucleotide
delivery-enhancing polypeptide is a histone protein, or a
polypeptide or peptide fragment, derivative, analog, or conjugate
thereof. Within these embodiments, the siNA is admixed, complexed
or conjugated with one or more full length histone proteins or
polypeptides corresponding at least in part to a partial sequence
of a histone protein, for example of one or more of the following
histones: histone H1, histone H2A, histone H2B, histone H3 or
histone H4, or one or more polypeptide fragments or derivatives
thereof comprising at least a partial sequence of a histone
protein, typically at least 5-10 or 10-20 contiguous residues of a
native histone protein. In more detailed embodiments, the
siRNA/histone mixture, complex or conjugate is substantially free
of amphipathic compounds. In other detailed embodiments, the siNA
that is admixed, complexed, or conjugated with the histone protein
or polypeptide will comprise a double-stranded double-stranded RNA,
for example a double-stranded RNA that has 30 or fewer nucleotides,
and is a short interfering RNA (siRNA). In exemplary embodiments,
the histone polynucleotide delivery-enhancing polypeptide comprises
a fragment of histone H2B, as exemplified by the polynucleotide
delivery-enhancing polypeptide designated PN73 described herein
below. In yet additional detailed embodiments, the polynucleotide
delivery-enhancing polypeptide may be pegylated to improve
stability and/or efficacy, particularly in the context of in vivo
administration.
[0016] Within additional embodiments of the invention, the
polynucleotide delivery-enhancing polypeptide is selected or
rationally designed to comprise an amphipathic amino acid sequence.
For example, useful polynucleotide delivery-enhancing polypeptides
may be selected which comprise a plurality of non-polar or
hydrophobic amino acid residues that form a hydrophobic sequence
domain or motif, linked to a plurality of charged amino acid
residues that form a charged sequence domain or motif, yielding an
amphipathic peptide.
[0017] In other embodiments, the polynucleotide delivery-enhancing
polypeptide is selected to comprise a protein transduction domain
or motif, and a fusogenic peptide domain or motif. A protein
transduction domain is a peptide sequence that is able to insert
into and preferably transit through the membrane of cells. A
fusogenic peptide is a peptide that is able destabilize a lipid
membrane, for example a plasma membrane or membrane surrounding an
endosome, which may be enhanced at low pH. Exemplary fusogenic
domains or motifs are found in a broad diversity of viral fusion
proteins and in other proteins, for example fibroblast growth
factor 4 (FGF4).
[0018] To rationally design polynucleotide delivery-enhancing
polypeptides of the invention, a protein transduction domain is
employed as a motif that will facilitate entry of the nucleic acid
into a cell through the plasma membrane. In certain embodiments,
the transported nucleic acid will be encapsulated in an endosome.
The interior of endosomes has a low pH resulting in the fusogenic
peptide motif destabilizing the membrane of the endosome. The
destabilization and breakdown of the endosome membrane allows for
the release of the siNA into the cytoplasm where the siNA can
associate with a RISC complex and be directed to its target
mRNA.
[0019] Examples of protein transduction domains for optional
incorporation into polynucleotide delivery-enhancing polypeptides
of the invention include: 1. TAT protein transduction domain (PTD)
(SEQ ID NO: 1) KRRQRRR; 2. Penetratin PTD (SEQ ID NO: 2)
RQIKIWFQNRRMKWKK; 3. VP22 PTD (SEQ ID NO: 3)
DAATATRGRSAASRPTERPRAPARSASRPRRPVD; 4. Kaposi FGF signal sequences
(SEQ ID NO: 4) AAVALLPAVLLALLAP, and SEQ ID NO: 5)
AAVLLPVLLPVLLAAP; 5. Human .beta.3 integrin signal sequence (SEQ ID
NO: 6) VTVLALGALAGVGVG; 6. gp41 fusion sequence (SEQ ID NO: 7)
GALFLGWLGAAGSTMGA; 7. Caiman crocodylus Ig(v) light chain (SEQ ID
NO: 8) MGLGLHLLVLAAALQGA; 8. hCT-derived peptide (SEQ ID NO: 9)
LGTYTQDFNKFHTFPQTAIGVGAP; 9. Transportan (SEQ ID NO: 10)
GWTLNSAGYLLKINLKALAALAKKIL; 10. Loligomer (SEQ ID NO: 11)
TPPKKKRKVEDPKKKK; 11. Arginine peptide (SEQ ID NO: 12) RRRRRRR; and
12. Amphiphilic model peptide (SEQ ID NO: 13)
KLALKLALKALKAALKLA.
[0020] Examples of viral fusion peptides fusogenic domains for
optional incorporation into polynucleotide delivery-enhancing
polypeptides of the invention include: 1. Influenza HA2 (SEQ ID NO:
14) GLFGAIAGFIENGWEG; 2. Sendai F1 (SEQ ID NO: 15)
FFGAVIGTIALGVATA; 3. Respiratory Syncytial virus F1 (SEQ ID NO: 16)
FLGFLLGVGSAIASGV; 4. HIV gp41 (SEQ ID NO: 17) GVFVLGFLGFLATAGS; and
5. Ebola GP2 (SEQ ID NO: 18) GAAIGLAWIPYFGPAA.
[0021] Within yet additional embodiments of the invention,
polynucleotide delivery-enhancing polypeptides are provided that
incorporate a DNA-binding domain or motif which facilitates
polypeptide-siNA complex formation and/or enhances delivery of
siNAs within the methods and compositions of the invention.
Exemplary DNA binding domains in this context include various "zinc
finger" domains as described for DNA-binding regulatory proteins
and other proteins identified in Table 1, below (see, e.g., Simpson
et al., J. Biol. Chem. 278:28011-28018, 2003). TABLE-US-00001 TABLE
1 Exemplary zinc finger motifs of different DNA-binding proteins
C.sub.2H.sub.2 Zinc finger motif ....|....| ....|....| ....|....|
....|....| ....|....| ....|....| 665 675 685 695 705 715 Sp1
ACTCPYCKDS EGRGSG---- DPGKKKQHIC HIQGCGKVYG KTSHLRAHLR WHTGERPFMC
Sp2 ACTCPNCKDG EKRS------ GEQGKKKHVC HIPDCGKTFR KTSLLRAHVR
LHTGERPFVC Sp3 ACTCPNCKEG GGRGTN---- -LGKKKQHIC HIPGCGKVYG
KTSHLRAHLR WHSGERPFVC Sp4 ACSCPNCREG EGRGSN---- EPGKKKQHIC
HIEGCGKVYG KTSHLRAHLR WHTGERPFIC DrosBtd RCTCPNCTNE MSGLPPIVGP
DERGRKQHIC HIPGCERLYG KASHLKTHLR WHTGERPFLC DrosSp TCDCPNCQEA
ERLGPAGV-- HLRKKNIHSC HIPGCGKVYG KTSHLKAHLR WHTGERPFVC CeT22C8.5
RCTCPNCKAI KHG------- DRGSQBTHLC SVPGCGKTYK KTSHLRAHLR KHTGDRPFVC
Y40B1A.4 Prosite pattern C--x(2,4) --C--x(12)--H--x(3)--H *The
table demonstrates a conservative zinc fingerer motif for double
strand DNA binding which is characterized by the C--x(2,4)
--C--x(12)--H--x(3)--H motif pattern, which itself can be used to
select and design additional polynucleotide delivery-enhancing
polypeptides according to the invention. **The sequences shown in
Table 1, for Sp1, Sp2, Sp3, Sp4, DrosBtd, DrosSp, CeT22C8.5, and
Y4pB1A.4, are herein assigned SEQ ID NO:s 19, 20, 21, 22, 23, 24,
25, and 26, respectively.
[0022] Alternative DNA binding domains useful for constructing
polynucleotide delivery-enhancing polypeptides of the invention
include, for example, portions of the HIV Tat protein sequence
(see, Examples, below).
[0023] Within exemplary embodiments of the invention described
herein below, polynucleotide delivery-enhancing polypeptides may be
rationally designed and constructed by combining any of the
foregoing structural elements, domains or motifs into a single
polypeptide effective to mediate enhanced delivery of siNAs into
target cells. For example, a protein transduction domain of the TAT
polypeptide was fused to the N-terminal 20 amino acids of the
influenza virus hemagglutinin protein, termed HA2, to yield one
exemplary polynucleotide delivery-enhancing polypeptide herein.
Various other polynucleotide delivery-enhancing polypeptide
constructs are provided in the instant disclosure, evincing that
the concepts of the invention are broadly applicable to create and
use a diverse assemblage of effective polynucleotide
delivery-enhancing polypeptides for enhancing siNA delivery.
[0024] Yet additional exemplary polynucleotide delivery-enhancing
polypeptides within the invention may be selected from the
following peptides: WWETWKPFQCRICMRNFSTRQARRNHRRRHR (SEQ ID NO:
27); GKINLKALAALAKKIL (SEQ ID NO: 28), RVIRVWFQNKRCKDKK (SEQ ID NO:
29), GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO: 30),
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 31), Poly Lys-Trp, 4:1, MW
20,000-50,000; and Poly Orn-Trp, 4:1, MW 20,000-50,000. Additional
polynucleotide delivery-enhancing polypeptides that are useful
within the compositions and methods herein comprise all or part of
the mellitin protein sequence.
[0025] Still other exemplary polynucleotide delivery-enhancing
polypeptides are identified in the examples below. Any one or
combination of these peptides may be selected or combined to yield
effective polynucleotide delivery-enhancing polypeptide reagents to
induce or facilitate intracellular delivery of siNAs within the
methods and compositions of the invention.
[0026] In more detailed aspects of the invention, the mixture,
complex or conjugate comprising a siRNA and a polynucleotide
delivery-enhancing polypeptide can be optionally combined with
(e.g., admixed or complexed with) a cationic lipid, such as
LIPOFECTIN.RTM.. In this context it is unexpectedly disclosed
herein that polynucleotide delivery-enhancing polypeptides
complexed or conjugated to a siRNA alone will effectuate delivery
of the siNA sufficient to mediate gene silencing by RNAi. However,
it is further unexpectedly disclosed herein that a
siRNA/polynucleotide delivery-enhancing polypeptide complex or
conjugate will exhibit even greater activity for mediating siNA
delivery and gene silencing when admixed or complexed with a
cationic lipid, such as lipofectin. To produce these compositions
comprised of a polynucleotide delivery-enhancing polypeptide, siRNA
and a cationic lipid, the siRNA and peptide may be mixed together
first in a suitable medium such as a cell culture medium, after
which the cationic lipid is added to the mixture to form a
siRNA/delivery peptide/cationic lipid composition. Optionally, the
peptide and cationic lipid can be mixed together first in a
suitable medium such as a cell culture medium, whereafter the siRNA
can be added to form the siRNA/delivery peptide/cationic lipid
composition.
[0027] Examples of useful cationic lipids within these aspects of
the invention include
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride,
1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane,
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide, and
dimethyldioctadecylammonium bromide,
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini-
um trifluoracetate,
1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamid,
5-carboxyspermylglycine dioctadecylamide, tetramethyltetrapalmitoyl
spermine, tetramethyltetraoleyl spermine, tetramethyltetralauryl
spermine, tetramethyltetramyristyl spermine and tetramethyldioleyl
spermine. DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl
ammonium chloride), DOTAP
(1,2-bis(oleoyloxy)-3,3-(trimethylammonium)propane), DMRIE
(1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide)
or DDAB (dimethyl dioctadecyl ammonium bromide). Polyvalent
cationic lipids include lipospermines, specifically DOSPA
(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-
ium trifluoro-acetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxy
spermyl)-propyl-amid, and the di- and tetra-alkyl-tetra-methyl
spermines, including but not limited to TMTPS
(tetramethyltetrapalmitoyl spermine), TMTOS (tetramethyltetraoleyl
spermine), TMTLS (tetramethlytetralauryl spermine), TMTMS
(tetramethyltetramyristyl spermine) and TMDOS (tetramethyldioleyl
spermine) DOGS (dioctadecyl-amidoglycylspermine (TRANSFECTAM.RTM.).
Other useful cationic lipids are described, for example, in U.S.
Pat. No. 6,733,777; U.S. Pat. No 6,376,248; U.S. Pat. No.
5,736,392; U.S. Pat. No. 5,686,958; U.S. Pat. No. 5,334,761 and
U.S. Pat. No. 5,459,127.
[0028] Cationic lipids are optionally combined with non-cationic
lipids, particularly neutral lipids, for example lipids such as
DOPE (dioleoylphosphatidylethanolamine), DPhPE
(diphytanoylphosphatidylethanolamine) or cholesterol. A cationic
lipid composition composed of a 3:1 (w/w) mixture of DOSPA and DOPE
or a 1:1 (w/w) mixture of DOTMA and DOPE (LIPOFECTIN.RTM.,
Invitrogen) are generally useful in transfecting compositions of
this invention. Preferred transfection compositions are those which
induce substantial transfection of a higher eukaryotic cell
line.
[0029] In exemplary embodiments, the instant invention features
compositions comprising a small nucleic acid molecule, such as
short interfering nucleic acid (siNA), a short interfering RNA
(siRNA), a double-stranded RNA (dsRNA), micro-RNA (mRNA), or a
short hairpin RNA (shRNA), admixed or complexed with, or conjugated
to, a polynucleotide delivery-enhancing polypeptide.
[0030] As used herein, the term "short interfering nucleic acid",
"siNA", "short interfering RNA", "siRNA", "short interfering
nucleic acid molecule", "short interfering oligonucleotide
molecule", or "chemically-modified short interfering nucleic acid
molecule", refers 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. Within exemplary 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 nucleic acid
molecule for downregulating 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
nucleic acid sequence or portion thereof.
[0031] "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), and which is not
unacceptably toxic in target cells. The length of useful siNAs
within the invention will in certain embodiments be optimized at a
length of approximately 21 to 23 bp long. However, there is no
particular limitation in 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 following the time of delivery to yield a siNA that is active
within the cell to mediate gene silencing. Thus, in certain
embodiments, useful siNAs within the invention will have a
precursor length, for example, of approximately 100-200 base pairs,
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, 15 to 35 bp, or about 21 to 30 bp in length.
[0032] In certain embodiments of the invention, as noted above,
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. 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.
[0033] In mammalian cells, dsRNAs longer than 30 base pairs can
activate the dsRNA-dependent kinase PKR and 2'-5'-oligoadenylate
synthetase, normally induced by interferon. The activated PKR
inhibits general translation by phosphorylation of the translation
factor eukaryotic initiation factor 2a (eIF2a), while
2'-5'-oligoadenylate synthetase causes nonspecific mRNA degradation
via activation of RNase L. By virtue of their small size (referring
particularly to non-precursor forms), usually less than 30 base
pairs, and most commonly between about 17-19, 19-21, or 21-23 base
pairs, the siNAs of the present invention avoid activation of the
interferon response.
[0034] In contrast to the nonspecific effect of long dsRNA, siRNA
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.
[0035] RISC mediates cleavage of single stranded RNA having
sequence complementary to the antisense strand of the siRNA duplex.
Cleavage of the target RNA takes place in the middle of the region
complementary to the antisense strand of the siRNA duplex. Studies
have shown that 21 nucleotide siRNA duplexes are most active when
containing two nucleotide 3'-overhangs. Furthermore, complete
substitution of one or both siRNA strands with 2'-deoxy (2'-H) or
2'-O-methyl nucleotides abolishes RNAi activity, whereas
substitution of the 3'-terminal siRNA overhang nucleotides with
deoxy nucleotides (2'-H) has been reported to be tolerated.
[0036] Studies have shown that replacing the 3'-overhanging
segments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangs
with deoxyribonucleotides does not have an adverse effect on RNAi
activity. Replacing up to 4 nucleotides on each end of the siRNA
with deoxyribonucleotides has been reported to be well tolerated
whereas complete substitution with deoxyribonucleotides results in
no RNAi activity.
[0037] 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 bp long. Within
exemplary embodiments, double-stranded portions of siNAs, in which
two strands pair up, are not limited to completely paired
nucleotide segments, and may contain nonpairing portions due to
mismatch (the corresponding nucleotides are not complementary),
bulge (lacking in the corresponding complementary nucleotide on one
strand), overhang, and the like. Nonpairing portions can be
contained to the extent that they do not interfere with siNA
formation. In more detailed embodiments, a "bulge" may comprise 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 cause are also
contemplated. Furthermore, in the present invention the
double-stranded region of siNAs in which two strands pair up may
contain both bulge and mismatched portions in the approximate
numerical ranges specified.
[0038] The terminal structure of siNAs of the 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 only to the 3' overhang
as reported by others. On the contrary, the 5' overhanging
structure may be included as long as it is capable of inducing a
gene silencing effect such as by RNAi. In addition, the number of
overhanging nucleotides is not limited to reported limits of 2 or 3
nucleotides, but can be any number as long as the overhang does not
impair gene silencing activity of the siNA. For example, overhangs
may comprise from about 1 to 8 nucleotides, more often from about 2
to 4 nucleotides. The total length of siNAs having cohesive end
structure is expressed as the sum of the length of the paired
double-stranded portion and that of a pair comprising overhanging
single-strands at both ends. For example, in the exemplary case of
a 19 bp double-stranded RNA with 4 nucleotide overhangs at both
ends, the total length is expressed as 23 bp. Furthermore, since
the overhanging sequence may have low specificity to a target gene,
it is not necessarily complementary (antisense) or identical
(sense) to the target gene sequence. Furthermore, as long as the
siNA is able to maintain its gene silencing effect on the target
gene, it may contain low molecular weight structure (for example a
natural RNA molecule such as tRNA, rRNA or viral RNA, or an
artificial RNA molecule), for example, in the overhanging portion
at one end.
[0039] In addition, 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, e.g., a linker RNA. The length of the double-stranded region
(stem-loop portion) can be, for example, 15 to 49 bp, often 15 to
35 bp, and more commonly about 21 to 30 bp 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, 15 to 35 bp, or about 21 to 30
bp long. When linker segments are employed, there is no particular
limitation in the length of the linker as long as it does not
hinder pairing of the stem portion. For example, for stable pairing
of the stem portion and suppression of recombination between DNAs
coding for this portion, the linker portion may have a clover-leaf
tRNA structure. Even if the linker has a length that would hinder
pairing of the stem portion, it is possible, for example, to
construct the linker portion to include introns so that the introns
are excised during processing of a precursor RNA into mature RNA,
thereby allowing pairing of the stem portion. In the case of a
stem-loop siRNA, either end (head or tail) of RNA with no loop
structure may have a low molecular weight RNA. As described above,
these low molecular weight RNAs may include a natural RNA molecule,
such as tRNA, rRNA or viral RNA, or an artificial RNA molecule.
[0040] The siNA can also comprise a single stranded polynucleotide
having nucleotide sequence complementary to nucleotide sequence in
a target nucleic acid molecule or a portion thereof (for example,
where such siNA molecule does not require the presence within the
siNA molecule of nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof), wherein the single
stranded polynucleotide can further comprise a terminal phosphate
group, such as a 5'-phosphate (see for example Martinez et al.,
Cell., 110: 563-574 (2002) and Schwarz et al., Molecular Cell, 10:
537-568(2002), or 5',3'-diphosphate.
[0041] 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
certain embodiments, the short interfering nucleic acid molecules
of the invention lack 2'-hydroxy (2'-OH) containing nucleotides. In
certain 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 the
invention optionally do not include any ribonucleotides (e.g.,
nucleotides having a 2'-OH group). Such siNA molecules that do not
require the presence of ribonucleotides within the siNA molecule to
support RNAi can however have an attached linker or linkers or
other attached or associated groups, moieties, or chains containing
one or more nucleotides with 2'-OH groups. Optionally, siNA
molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40,
or 50% of the nucleotide positions.
[0042] As used herein, the term siNA is meant to be equivalent to
other terms used to describe nucleic acid molecules that are
capable of mediating sequence specific RNAi, for example short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(mRNA), short hairpin RNA (shRNA), short interfering
oligonucleotide, short interfering nucleic acid, short interfering
modified oligonucleotide, chemically-modified siRNA,
post-transcriptional gene silencing RNA (ptgsRNA), and others.
[0043] In other embodiments, siNA molecules for use within the
invention may 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
alternately non-covalently linked by ionic interactions, hydrogen
bonding, van der waals interactions, hydrophobic intercations,
and/or stacking interactions.
[0044] "Antisense RNA" is an RNA strand having a sequence
complementary to a target gene mRNA, and thought to induce RNAi by
binding to the target gene mRNA. "Sense RNA" has a sequence
complementary to the antisense RNA, and annealed to its
complementary antisense RNA to form siRNA. These antisense and
sense RNAs have been conventionally synthesized with an RNA
synthesizer.
[0045] As used herein, the term "RNAi construct" is a generic term
used throughout the specification to include small interfering RNAs
(siRNAs), hairpin RNAs, and other RNA species which can be cleaved
in vivo to form siRNAs. RNAi constructs herein also include
expression vectors (also referred to as RNAi expression vectors)
capable of giving rise to transcripts which form dsRNAs or hairpin
RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
Optionally, the siRNA include single strands or double strands of
siRNA.
[0046] An siHybrid molecule is a double-stranded nucleic acid that
has a similar function to siRNA. Instead of a double-stranded RNA
molecule, an siHybrid is comprised of an RNA strand and a DNA
strand. Preferably, the RNA strand is the antisense strand as that
is the strand that binds to the target mRNA. The siHybrid created
by the hybridization of the DNA and RNA strands have a hybridized
complementary portion and preferably at least one 3'overhanging
end.
[0047] 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).
[0048] Within additional embodiments, siNAs for intracellular
delivery according to the methods and compositions of the invention
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.
[0049] Non-limiting examples of chemical modifications that can be
made in an siNA include without limitation phosphorothioate
internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl
ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal
base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides,
and terminal glyceryl and/or inverted deoxy abasic residue
incorporation. These chemical modifications, when used in various
siNA constructs, are shown to preserve RNAi activity in cells while
at the same time, dramatically increasing the serum stability of
these compounds.
[0050] In a non-limiting example, the introduction of
chemically-modified nucleotides into nucleic acid molecules
provides a powerful tool in overcoming potential limitations of in
vivo stability and bioavailability inherent to native RNA molecules
that are delivered exogenously. For example, the use of
chemically-modified nucleic acid molecules can enable a lower dose
of a particular nucleic acid molecule for a given therapeutic
effect since chemically-modified nucleic acid molecules tend to
have a longer half-life in serum. Furthermore, certain chemical
modifications can improve the bioavailability of nucleic acid
molecules by targeting particular cells or tissues and/or improving
cellular uptake of the nucleic acid molecule. Therefore, even if
the activity of a chemically-modified nucleic acid molecule is
reduced as compared to a native nucleic acid molecule, for example,
when compared to an all-RNA nucleic acid molecule, the overall
activity of the modified nucleic acid molecule can be greater than
that of the native molecule due to improved stability and/or
delivery of the molecule. Unlike native unmodified siNA,
chemically-modified siNA can also minimize the possibility of
activating interferon activity in humans.
[0051] The siNA molecules described herein, the antisense region of
a siNA molecule of the invention can comprise a phosphorothioate
internucleotide linkage at the 3'-end of said antisense region. In
any of the embodiments of siNA molecules described herein, the
antisense region can comprise about one to about five
phosphorothioate internucleotide linkages at the 5'-end of said
antisense region. In any of the embodiments of siNA molecules
described herein, the 3'-terminal nucleotide overhangs of a siNA
molecule of the invention can comprise ribonucleotides or
deoxyribonucleotides that are chemically-modified at a nucleic acid
sugar, base, or backbone. In any of the embodiments of siNA
molecules described herein, the 3'-terminal nucleotide overhangs
can comprise one or more universal base ribonucleotides. In any of
the embodiments of siNA molecules described herein, the 3'-terminal
nucleotide overhangs can comprise one or more acyclic
nucleotides.
[0052] For example, in a non-limiting example, the invention
features a chemically-modified short interfering nucleic acid
(siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate
internucleotide linkages in one siNA strand. In yet another
embodiment, the invention features a chemically-modified short
interfering nucleic acid (siNA) individually having about 1, 2, 3,
4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in
both siNA strands. The phosphorothioate internucleotide linkages
can be present in one or both oligonucleotide strands of the siNA
duplex, for example in the sense strand, the antisense strand, or
both strands. The siNA molecules of the invention can comprise one
or more phosphorothioate internucleotide linkages at the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends of the sense strand, the
antisense strand, or both strands. For example, an exemplary siNA
molecule of the invention can comprise about 1 to about 5 or more
(e.g., about 1, 2, 3, 4, 5, or more) consecutive phosphorothioate
internucleotide linkages at the 5'-end of the sense strand, the
antisense strand, or both strands. In another non-limiting example,
an exemplary siNA molecule of the invention can comprise one or
more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
pyrimidine phosphorothioate internucleotide linkages in the sense
strand, the antisense strand, or both strands. In yet another
non-limiting example, an exemplary siNA molecule of the invention
can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more) purine phosphorothioate internucleotide linkages in
the sense strand, the antisense strand, or both strands.
[0053] An siNA molecule may be comprised of a circular nucleic acid
molecule, wherein the siNA is about 38 to about 70 (e.g., about 38,
40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about
18 to about 23 (e.g., 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.
[0054] A circular siNA molecule contains two loop motifs, wherein
one or both loop portions of the siNA molecule is biodegradable.
For example, a circular siNA molecule of the invention is designed
such that degradation of the loop portions of the siNA molecule in
vivo can generate a double-stranded siNA molecule with 3'-terminal
overhangs, such as 3'-terminal nucleotide overhangs comprising
about 2 nucleotides.
[0055] Modified nucleotides present in siNA molecules, preferably
in the antisense strand of the siNA molecules, but also optionally
in the sense and/or both antisense and sense strands, comprise
modified nucleotides having properties or characteristics similar
to naturally occurring ribonucleotides. For example, the invention
features siNA molecules including modified nucleotides having a
Northern conformation (e.g., Northern pseudorotation cycle, see for
example Saenger, Principles of Nucleic Acid Structure,
Springer-Verlag ed., 1984). As such, chemically modified
nucleotides present in the siNA molecules of the invention,
preferably in the antisense strand of the siNA molecules of the
invention, but also optionally in the sense and/or both antisense
and sense strands, are resistant to nuclease degradation while at
the same time maintaining the capacity to mediate RNAi.
Non-limiting examples of nucleotides having a northern
configuration include locked nucleic acid (LNA) nucleotides (e.g.,
2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides);
2'-methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl,
2'-deoxy-2'-fluoro micleotides. 2'-deoxy-2'-chloro nucleotides,
2'-azido nucleotides, and 2'-O-methyl nucleotides.
[0056] The sense strand of a double stranded siNA molecule may have
a terminal cap moiety such as an inverted deoxyabaisc moiety, at
the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand.
[0057] Non-limiting 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. In another embodiment, the
conjugate is covalently attached to the chemically-modified siNA
molecule via a biodegradable linker. In one embodiment, the
conjugate molecule is attached at the 3'-end of either the sense
strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In another embodiment, the
conjugate molecule is attached at the 5'-end of either the sense
strand, the antisense strand, or both strands of the
chemically-modified siNA molecule. In yet another embodiment, the
conjugate molecule is attached both the 3'-end and 5'-end of either
the sense strand, the antisense strand, or both strands of the
chemically-modified siNA molecule, or any combination thereof. In
one embodiment, a conjugate molecule of the invention comprises a
molecule that facilitates delivery of a chemically-modified siNA
molecule into a biological system, such as a cell. In another
embodiment, the conjugate molecule attached to the
chemically-modified siNA molecule is a poly ethylene 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 application Publication No. 20030130186, published
Jul. 10, 2003, and U.S. patent application Publication No.
20040110296, published Jun. 10, 2004. The type of conjugates used
and the extent of conjugation of siNA molecules of the invention
can be evaluated for improved pharmacokinetic profiles,
bioavailability, and/or stability of siNA constructs while at the
same time maintaining the ability of the siNA to mediate RNAi
activity. As such, one skilled in the art can screen siNA
constructs that are modified with various conjugates to determine
whether the siNA conjugate complex possesses improved properties
while maintaining the ability to mediate RNAi, for example in
animal models as are generally known in the art.
[0058] A siNA further may be further comprised of a nucleotide,
non-nucleotide, or mixed nucleotide/non-nucleotide linker that
joins the sense region of the siNA to the antisense region of the
siNA. In one embodiment, a nucleotide linker can be a linker of
>2 nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9,
or 10 nucleotides in length. In another embodiment, the nucleotide
linker can be a nucleic acid aptamer. By "aptamer" or "nucleic acid
aptamer" as used herein is meant a nucleic acid molecule that binds
specifically to a target molecule wherein the nucleic acid molecule
has sequence that comprises a sequence recognized by the target
molecule in its natural setting. Alternately, an aptamer can be a
nucleic acid molecule that binds to a target molecule where the
target molecule does not naturally bind to a nucleic acid. The
target molecule can be any molecule of interest. For example, the
aptamer can be used to bind to a ligand-binding domain of a
protein, thereby preventing interaction of the naturally occurring
ligand with the protein. This is a non-limiting example and those
in the art will recognize that other embodiments can be readily
generated using techniques generally known in the art. [See, for
example, Gold et al, 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) A non-nucleotide linker may be comprised of 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); Jschke et al., Tetrahedron Lett., 34:301 (1993); Ono
et al., Biochemistry, 30:9914 (1991); Arnold et al., International
Publication No. WO 89/02439; Usman et al., International
Publication No. WO 95/06731; Dudycz et al., International
Publication No. WO 95/11910 and Ferentz and Verdine, J. Am. Chem.
Soc., 113:4000 (1991). A "non-nucleotide" further means any group
or compound that can be incorporated into a nucleic acid chain in
the place of one or more nucleotide units, including either sugar
and/or phosphate substitutions, and allows the remaining bases to
exhibit their enzymatic activity. The group or compound can be
abasic in that it does not contain a commonly recognized nucleotide
base, such as adenosine, guanine, cytosine, uracil or thyrnine, for
example at the C1 position of the sugar.
[0059] The synthesis of a siNA molecule of the invention, which can
be chemically-modified, comprises: (a) synthesis of two
complementary strands of the siNA molecule; (b) annealing the two
complementary strands together under conditions suitable to obtain
a double-stranded siNA molecule. In another embodiment, synthesis
of the two complementary strands of the siNA molecule is by solid
phase oligonucleotide synthesis. In yet another embodiment,
synthesis of the two complementary strands of the siNA molecule is
by solid phase tandem oligonucleotide synthesis.
[0060] Oligonucleotides (e.g., certain modified oligonucleotides or
portions of oligonucleotides lacking ribonucleotides) are
synthesized using protocols known in the art, for example as
described in Caruthers et al., 1992, Methods in Enzymology 211,
3-19, Thompson et al., International PCT Publication No. WO
99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684,
Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al.,
1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.
6,001,311. Synthesis of RNA, including certain siNA molecules of
the invention, follows general procedures as described, for
example, in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845;
Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et
al., 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997,
Methods Mol. Bio., 74, 59.
[0061] 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 PCT Publication No WO 94/02595, further describes
general methods for delivery of enzymatic nucleic acid molecules.
These protocols can be utilized to supplement or complement
delivery of virtually any nucleic acid molecule contemplated within
the invention.
[0062] Nucleic acid molecules and polynucleotide delivery-enhancing
polypeptides can be administered to cells by a variety of methods
known to those of skill in the art, including, but not restricted
to, administration within formulations that comprise the siNA and
polynucleotide delivery-enhancing polypeptide alone, or that
further comprise one or more additional components, such as a
pharmaceutically acceptable carrier, diluent, excipient, adjuvant,
emulsifier, buffer, stabilizer, preservative, and the like. In
certain embodiments, the siNA and/or the polynucleotide
delivery-enhancing polypeptide can be encapsulated in liposomes,
administered by iontophoresis, or incorporated into other vehicles,
such as hydrogels, cyclodextrins, biodegradable nanocapsules,
bioadhesive microspheres, or proteinaceous vectors (see e.g.,
O'Hare and Normand, International PCT Publication No. WO 00/53722).
Alternatively, a nucleic acid/peptide/vehicle combination 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 PCT Publication No. WO 99/31262.
[0063] The compositions of the instant 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.
[0064] Thus within additional embodiments the 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
polynucleotide delivery-enhancing polypeptide, optionally
formulated with a pharmaceutically-acceptable carrier, such as a
diluent, stabilizer, buffer, and the like.
[0065] The present invention satisfies additional objects and
advantages by providing short interfering nucleic acid (siNA)
molecules that modulate expression of genes associated with a
particular disease state or other adverse condition in a subject.
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.
[0066] Within exemplary embodiments, the compositions and methods
of the invention are useful as therapeutic tools to regulate
expression of tumor necrosis factor-.alpha. (TNF-.alpha.) to treat
or prevent symptoms of rheumatoid arthritis (RA). In this context
the invention further provides compounds, compositions, and methods
useful for modulating expression and activity of TNF-.alpha. by RNA
interference (RNAi) using small nucleic acid molecules. In more
detailed embodiments, the invention provides small nucleic acid
molecules, such as short interfering nucleic acid (siNA), short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(mRNA), and short hairpin RNA (shRNA) molecules, and related
methods, that are effective for modulating expression of
TNF-.alpha. and/or TNF-.alpha. genes to prevent or alleviate
symptoms of RA in mammalian subjects. Within these and related
therapeutic compositions and methods, the use of
chemically-modified siNAs will often improve properties of the
modified siNAs in comparison to properties of native siNA
molecules, for example by providing increased resistance to
nuclease degradation in vivo, and/or through improved cellular
uptake. As can be readily determined according to the disclosure
herein, useful siNAs having multiple chemical modifications will
retain their RNAi activity. The siNA molecules of the instant
invention thus provide useful reagents and methods for a variety of
therapeutic, diagnostic, target validation, genomic discovery,
genetic engineering, and pharmacogenomic applications.
[0067] 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.
[0068] The siNA/polynucleotide delivery-enhancing polypeptide
mixtures of the invention can be administered in conjunction with
other standard treatments for a targeted disease condition, for
example in conjunction with therapeutic agents effective against
inflammatory diseases, such as RA or psoriasis. Examples of
combinatorially useful and effective agents in this context include
non-steroidal antiinflammatory drugs (NSAIDs), methotrexate, gold
compounds, D-penicillamine, the antimalarials, sulfasalazine,
glucocorticoids, and other TNF-.alpha. neutralizing agents such as
infliximab and entracept.
[0069] Negatively charged polynucleotides of the invention (e.g.,
RNA or DNA) can be administered to a patient by any standard means,
with or without stabilizers, buffers, and the like, to form a
pharmaceutical composition. When it is desired to use a liposome
delivery mechanism, standard protocols for formation of liposomes
can be followed. 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
compositions known in the art.
[0070] The present invention also includes pharmaceutically
acceptable formulations of the compositions described herein. These
formulations include salts of the above compounds, e.g., acid
addition salts, for example, salts of hydrochloric, hydrobromic,
acetic acid, and benzene sulfonic acid.
[0071] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., 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, 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.
[0072] 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. Each of these administration
routes expose the desired negatively charged polymers, e.g.,
nucleic acids, to an accessible diseased tissue. The rate of entry
of a drug into the circulation has been shown to be a function of
molecular weight or size. The use of a liposome or other drug
carrier comprising the compounds of the instant invention can
potentially localize the drug, for example, in certain tissue
types, such as the tissues of the reticular endothelial system
(RES). A liposome formulation that can facilitate the association
of drug with the surface of cells, such as, lymphocytes and
macrophages is also useful. This approach may provide enhanced
delivery of the drug to target cells by taking advantage of the
specificity of macrophage and lymphocyte immune recognition of
abnormal cells, such as cancer cells.
[0073] By "pharmaceutically acceptable formulation" is meant, a
composition or formulation that allows for the effective
distribution of the nucleic acid molecules of the instant invention
in the physical location most suitable for their desired activity.
Nonlimiting examples of agents suitable for formulation with the
nucleic acid molecules of the instant 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
non-limiting 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).
[0074] 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 edit. 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.
[0075] A pharmaceutically effective dose is that dose required to
prevent, inhibit the occurrence of, or treat (alleviate a symptom
to some extent, preferably all of the symptoms) a disease state.
The pharmaceutically effective dose depends on the type of disease,
the composition used, the route of administration, the type of
mammal being treated, the physical characteristics of the specific
mammal under consideration, concurrent medication, and other
factors that those skilled in the medical arts will recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body
weight/day of active ingredients is administered dependent upon
potency of the negatively charged polymer.
[0076] 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.
[0077] 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.
[0078] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, can also be present.
[0079] 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.
[0080] 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.
[0081] The siNAs can also be administered in the form of
suppositories, e.g., for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient that is solid at ordinary temperatures but
liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols.
[0082] 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.
[0083] 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 e.g., Eckstein et al., International Publication No.
WO 92/07065; 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 93/15187; and Rossi et al., International
Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold
et al., U.S. Pat. No. 6,300,074. 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.
[0084] There are several examples in the art describing sugar, base
and phosphate modifications that can be introduced into nucleic
acid molecules with significant enhancement in their nuclease
stability and efficacy. For example, oligonucleotides are modified
to enhance stability and/or enhance biological activity by
modification with nuclease resistant groups, for example, 2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide
base modifications. For a review see Usman and Cedergren,, 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 PCT No. WO 92/07065; 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 PCT No. WO 93/15187; Sproat, U.S.
Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270,
25702; Beigelman et al., International PCT publication No. WO
97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al.,
U.S. Pat. No. 5,627,053; Woolf et al., International PCT
Publication No. WO 98/13526; Thompson et al., 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.
[0085] 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.
[0086] 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, 331-417 (1995), and Mesmaeker et
al., Novel Backbone Replacements for Oligonucleotides, in
Carbohydrate Modifications in Antisense Research, ACS, 24-39
(1994).
[0087] 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., PCT
WO 94/02595 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 in liposomes, by iontophoresis, or by
incorporation into other vehicles, such as biodegradable polymers,
hydrogels, cyclodextrins (see for example Gonzalez et al.,
Bioconjugate Chem., 10: 1068-1074 (1999); Wang et al.,
International PCT publication Nos. WO 03/47518 and WO 03/46185),
poly(lactic-co-glycolic)ac- id (PLGA) and PLCA microspheres (see
for example U.S. Pat. No. 6,447,796 and U.S. patent application
Publication No. US 2002130430), biodegradable nanocapsules, and
bioadhesive microspheres, or by proteinaceous vectors (O'Hare and
Normand, International PCT Publication No. WO 00/53722).
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 PCT
Publication No. WO 99/31262. 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.
[0088] The term "ligand" refers to any compound or molecule, such
as a drug, peptide, hormone, or neurotransmitter, that is capable
of interacting with another compound, such as a receptor, either
directly or indirectly. The receptor that interacts with a ligand
can be present on the surface of a cell or can alternately be an
intercullular receptor. Interaction of the ligand with the receptor
can result in a biochemical reaction, or can simply be a physical
interaction or association.
[0089] By "asymmetric hairpin" as used herein is meant a linear
siNA molecule comprising an antisense region, a loop portion that
can comprise nucleotides or non-nucleotides, and a sense region
that comprises fewer nucleotides than the antisense region to the
extent that the sense region has enough complementary nucleotides
to base pair with the antisense region and form a duplex with loop.
For example, an asymmetric hairpin siNA molecule of the invention
can comprise an antisense region having length sufficient to
mediate RNAi in a T-cell (e.g. about 19 to about 22 (e.g., about
19, 20, 21, or 22) nucleotides) and a loop region comprising about
4 to about 8 (e.g., about 4, 5, 6, 7, or 8) nucleotides, and a
sense region having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are
complementary to the antisense region. The asymmetric hairpin siNA
molecule can also comprise a 5'-terminal phosphate group that can
be chemically modified. The loop portion of the asymmetric hairpin
siNA molecule can comprise nucleotides, non-nucleotides, linker
molecules, or conjugate molecules as described herein.
[0090] By "asymmetric duplex" as used herein is meant a siNA
molecule having two separate strands comprising a sense region and
an antisense region, wherein the sense region comprises fewer
nucleotides than the antisense region to the extent that the sense
region has enough complementary nucleotides to base pair with the
antisense region and form a duplex. For example, an asymmetric
duplex siNA molecule of the invention can comprise an antisense
region having length sufficient to mediate RNAi in a T-cell (e.g.
about 19 to about 22 (e.g. about 19, 20, 21, or 22) nucleotides)
and a sense region having about 3 to about 18 (e.g., about
3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, or 18) nucleotides
that are complementary to the antisense region.
[0091] By "modulate gene expression" is meant that the expression
of a target gene is upregulated or downregulated, 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. Modulation of gene expression
can be determined also be the presence, quantity, or activity of
one or more proteins or protein subunits encoded by the target gene
that is up regulated or down regulated, such that expression,
level, or activity of the subject protein or subunit is greater
than or less than that which is observed in the absence of the
modulator (e.g., a siRNA). For example, the term "modulate" can
mean "inhibit," but the use of the word "modulate" is not limited
to this definition.
[0092] By "inhibit", "down-regulate", or "reduce" expression, it is
meant that the expression of the gene, or level of RNA molecules or
equivalent RNA molecules encoding one or more proteins or protein
subunits, or 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. In one embodiment, inhibition, down-regulation or
reduction with an siNA molecule is below that level observed in the
presence of an inactive or attenuated molecule. In another
embodiment, inhibition, down-regulation, or reduction with siNA
molecules is below that level observed in the presence of, for
example, an siNA molecule with scrambled sequence or with
mismatches. In another embodiment, inhibition, down-regulation, or
reduction of gene expression with a nucleic acid molecule of the
instant invention is greater in the presence of the nucleic acid
molecule than in its absence.
[0093] Gene "silencing" refers to partial or complete
loss-of-function through targeted inhibition of gene expression in
a cell and may also be referred to as "knock down". Depending on
the circumstances and the biological problem to be addressed, it
may be preferable to partially reduce gene expression.
Alternatively, it might be desirable to reduce gene expression as
much as possible. The extent of silencing may be determined by
methods known in the art, some of which are summarized in
International Publication No. WO 99/32619. Depending on the assay,
quantitation of gene expression permits detection of various
amounts of inhibition that may be desired in certain embodiments of
the invention, including prophylactic and therapeutic methods,
which will be capable of knocking down target gene expression, in
terms of mRNA levels or protein levels or activity, for example, by
equal to or greater than 10%, 30%, 50%, 75% 90%, 95% or 99% of
baseline (i.e., normal) or other control levels, including elevated
expression levels as may be associated with particular disease
states or other conditions targeted for therapy.
[0094] The phrase "inhibiting expression of a target gene" refers
to the ability of a siNA of the invention to initiate gene
silencing of the target gene. To examine the extent of gene
silencing, samples or assays of the organism of interest or cells
in culture expressing a particular construct are compared to
control samples lacking expression of the construct. Control
samples (lacking construct expression) are assigned a relative
value of 100%. Inhibition of expression of a target gene is
achieved when the test value relative to the control is about 90%,
often 50%, and in certain embodiments 25-0%. Suitable assays
include, e.g., examination of protein or mRNA levels using
techniques known to those of skill in the art such as dot blots,
northern blots, in situ hybridization, ELISA, immunoprecipitation,
enzyme function, as well as phenotypic assays known to those of
skill in the art.
[0095] By "subject" is meant an organism, tissue, or cell, which
may include an organism as the subject or as a donor or recipient
of explanted cells or the cells that are themselves subjects for
siNA delivery. "Subject" therefore may refers to an organism,
organ, tissue, or cell, including in vitro or ex vivo organ, tissue
or cellular subjects, to which the nucleic acid molecules of the
invention can be administered and enhanced by polynucleotide
delivery-enhancing polypeptides described herein. Exemplary
subjects include mammalian individuals or cells, for example human
patients or cells.
[0096] As used herein "cell" is used in its usual biological sense,
and does not refer to an entire multicellular organism, e.g.,
specifically does not refer to a human. The cell can be present in
an organism, e.g., birds, plants and mammals such as humans, cows,
sheep, apes, monkeys, swine, dogs, and cats. The cell can be
prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian
or plant cell). The cell can be of somatic or germ line origin,
totipotent or pluripotent, dividing or non-dividing. The cell can
also be derived from or can comprise a gamete or embryo, a stem
cell, or a fully differentiated cell.
[0097] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0098] By "comprising" is meant including, but not limited to,
whatever follows the word "comprising." Thus, use of the term
"comprising" indicates that the listed elements are required or
mandatory, but that other elements are optional and may or may not
be present. By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of." Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they affect the
activity or action of the listed elements.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] By "antisense region" is meant a nucleotide sequence of a
siNA molecule having complementarity to a target nucleic acid
sequence. In addition, the antisense region of a siNA molecule can
optionally comprise a nucleic acid sequence having complementarity
to a sense region of the siNA molecule.
[0103] By "target nucleic acid" is meant any nucleic acid sequence
whose expression or activity is to be modulated. The target nucleic
acid can be DNA or RNA.
[0104] By "complementarity" is meant that a nucleic acid can form
hydrogen bond(s) with another nucleic acid sequence by either
traditional Watson-Crick or other non-traditional types. In
reference to the nucleic molecules of the present invention, the
binding free energy for a nucleic acid molecule with its
complementary sequence is sufficient to allow the relevant function
of the nucleic acid to proceed, e.g., RNAi activity. Determination
of binding free energies for nucleic acid molecules is well known
in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol.
LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.
109:3783-3785). A percent complementarity indicates the percentage
of contiguous residues in a nucleic acid molecule that can form
hydrogen bonds (e.g., Watson-Crick base pairing) with a second
nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out
of a total of 10 nucleotides in the first oligonuelcotide being
based paired to a second nucleic acid sequence having 10
nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100%
complementary respectively). "Perfectly complementary" means that
all the contiguous residues of a nucleic acid sequence will
hydrogen bond with the same number of contiguous residues in a
second nucleic acid sequence.
[0105] The term "universal base" as used herein refers to
nucleotide base analogs that form base pairs with each of the
natural DNA/RNA bases with little discrimination between them.
Non-limiting examples of universal bases include C-phenyl,
C-naphthyl and other aromatic derivatives, inosine, azole
carboxamides, and nitroazole derivatives such as 3-nitropyrrole,
4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art
(see for example Loakes, 2001, Nucleic Acids Research, 29,
2437-2447).
[0106] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of
the ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.
[0107] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example enzymatic
degradation or chemical degradation.
[0108] The term "biologically active molecule" as used herein,
refers to compounds or molecules that are capable of eliciting or
modifying a biological response in a system. Non-limiting examples
of biologically active siNA molecules either alone or in
combination with other molecules contemplated by the instant
invention include therapeutically active molecules such as
antibodies, cholesterol, hormones, antivirals, peptides, proteins,
chemotherapeutics, small molecules, vitamins, co-factors,
nucleosides, nucleotides, oligonucleotides, enzymatic nucleic
acids, antisense nucleic acids, triplex forming oligonucleotides,
2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and
analogs thereof. Biologically active molecules of the invention
also include molecules capable of modulating the pharmacokinetics
and/or pharmacodynamics of other biologically active molecules, for
example, lipids and polymers such as polyamines, polyamides,
polyethylene glycol and other polyethers.
[0109] The term "phospholipid" as used herein, refers to a
hydrophobic molecule comprising at least one phosphorus group. For
example, a phospholipid can comprise a phosphorus-containing group
and saturated or unsaturated alkyl group, optionally substituted
with OH, COOH, oxo, amine, or substituted or unsubstituted aryl
groups.
[0110] By "cap structure" is meant chemical modifications, which
have been incorporated at either terminus of the oligonucleotide
(see, for example, Adamic et al., U.S. Pat. No. 5,998,203,
incorporated by reference herein). These terminal modifications
protect the nucleic acid molecule from exonuclease degradation, and
may help in delivery and/or localization within a cell. The cap may
be present at the 5'-terminus (5'-cap) or at the 3'-terminal
(3'-cap) or may be present on both termini. In non-limiting
examples, the 5'-cap includes, but is not limited to, glyceryl,
inverted deoxy abasic residue (moiety); 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide;
L-nucleotides; alpha-nucleotides; modified base nucleotide;
phosphorodithioate linkage; threo-pentofuranosyl nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl
nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted
nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted
nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl
phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate;
or bridging or non-bridging methylphosphonate moiety.
[0111] Non-limiting examples of the 3'-cap include, but are not
limited to, glyceryl, inverted deoxy abasic residue (moiety),
4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide;
4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl
phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate;
6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl
phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;
alpha-nucleotide; modified base nucleotide; phosphorodithioate;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide;
3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,
5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety;
5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate;
5'-amino; bridging and/or non-bridging 5'-phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non
bridging methylphosphonate and 5'-mercapto moieties (for more
details see Beaucage and lyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0112] 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.
[0113] By "nucleotide" as used herein is as recognized in the art
to include natural bases (standard), and modified bases well known
in the art. Such bases are generally located at the 1' position of
a nucleotide sugar moiety. Nucleotides generally comprise a base,
sugar and a phosphate group. The nucleotides can be unmodified or
modified at the sugar, phosphate and/or base moiety, (also referred
to interchangeably as nucleotide analogs, modified nucleotides,
non-natural nucleotides, non-standard nucleotides and other; see,
for example, Usman and McSwiggen, supra; Eckstein et al.,
International PCT Publication No. WO 92/07065; Usman et al,
International PCT Publication No. WO 93/15187; Uhlman & Peyman,
supra, all are hereby incorporated by reference herein). There are
several examples of modified nucleic acid bases known in the art as
summarized by Limbach et al, 1994, Nucleic Acids Res. 22, 2183.
Some of the non-limiting examples of base modifications that can be
introduced into nucleic acid molecules include, inosine, purine,
pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,
2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,
naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),
5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,
5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
6-methyluridine), propyne, and others (Burgin et al., 1996,
Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modified
bases" in this aspect is meant nucleotide bases other than adenine,
guanine, cytosine and uracil at 1' position or their
equivalents.
[0114] By "target site" is meant a sequence within a target 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.
[0115] By "detectable level of cleavage" is meant cleavage of
target RNA (and formation of cleaved product RNAs) to an extent
sufficient to discern cleavage products above the background of
RNAs produced by random degradation of the target RNA. Production
of cleavage products from 1-5% of the target RNA is sufficient to
detect above the background for most methods of detection.
[0116] By "biological system" is meant, material, in a purified or
unpurified form, from biological sources, including but not limited
to human, animal, plant, insect, bacterial, viral or other sources,
wherein the system comprises the components required for RNAi
acitivity. The term "biological system" includes, for example, a
cell, tissue, or organism, or extract thereof. The term biological
system also includes reconstituted RNAi systems that can be used in
an in vitro setting.
[0117] The term "biodegradable linker" as used herein, refers to a
nucleic acid or non-nucleic acid linker molecule that is designed
as a biodegradable linker to connect one molecule to another
molecule, for example, a biologically active molecule to a siNA
molecule of the invention or the sense and antisense strands of a
siNA molecule of the invention. The biodegradable linker is
designed such that its stability can be modulated for a particular
purpose, such as delivery to a particular tissue or cell type. The
stability of a nucleic acid-based biodegradable linker molecule can
be modulated by using various chemistries, for example combinations
of ribonucleotides, deoxyribonucleotides, and chemically-modified
nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino,
2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified
nucleotides. The biodegradable nucleic acid linker molecule can be
a dimer, trimer, tetramer or longer nucleic acid molecule, for
example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or
can comprise a single nucleotide with a phosphorus-based linkage,
for example, a phosphoramidate or phosphodiester linkage. The
biodegradable nucleic acid linker molecule can also comprise
nucleic acid backbone, nucleic acid sugar, or nucleic acid base
modifications.
[0118] By "abasic" is meant sugar moieties lacking a base or having
other chemical groups in place of a base at the 1' position, see
for example Adamic et al., U.S. Pat. No. 5,998,203.
[0119] By "unmodified nucleoside" is meant one of the bases
adenine, cytosine, guanine, thymine, or uracil joined to the 1'
carbon of .beta.-D-ribo-furanose.
[0120] By "modified nucleoside" is meant any nucleotide base which
contains a modification in the chemical structure of an unmodified
nucleotide base, sugar and/or phosphate. Non-limiting examples of
modified nucleotides are shown by Formulae I-VII and/or other
modifications described herein.
[0121] In connection with 2'-modified nucleotides as described for
the present invention, by "amino" is meant 2'-NH.sub.2 or
2'-O--NH.sub.2, which can be modified or unmodified. Such modified
groups are described, for example, in Eckstein et al., U.S. Pat.
No. 5,672,695 and Matulic-Adanic et al., U.S. Pat. No.
6,248,878.
[0122] 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 polynucleotide
delivery-enhancing polypeptide, or both. The attached PEG can be
any molecular weight, preferably from about 2,000 to about 50,000
daltons (Da).
[0123] The sense region can be connected to the antisense region
via a linker molecule, such as a polynucleotide linker or a
non-nucleotide linker.
[0124] "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.
[0125] "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).
[0126] "Large double-stranded RNA" refers to any double-stranded
RNA having a size greater than about 40 base pairs (bp) for
example, larger than 100 bp or more particularly larger than 300
bp. The sequence of a large dsRNA may represent a segment of a mRNA
or the entire mRNA. The maximum size of the large dsRNA is not
limited herein. The double-stranded RNA may include modified bases
where the modification may be to the phosphate sugar backbone or to
the nucleoside. Such modifications may include a nitrogen or sulfur
heteroatom or any other modification known in the art.
[0127] 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.
[0128] "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.
[0129] "One or more dsRNAs" refers to dsRNAs that differ from each
other on the basis of sequence.
[0130] "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.
[0131] In this specification and the appended claims, the singular
forms of "a", "an" and "the" include plural reference unless the
context clearly dictates otherwise.
EXAMPLE 1
Production and Characterization of Compositions Comprising a siRNA
Complexed with a Polynucleotide Delivery-Enhancing Polypeptide
[0132] To form complexes between candidate siRNAs and
polynucleotide delivery-enhancing polypeptides of the invention, an
adequate amount of siRNA is combined with a pre-determined amount
of polynucleotide delivery-enhancing polypeptide, for example in
Opti-MEM.RTM. cell medium (Invitrogen), in defined ratios and
incubated at room temperature for about 10-30 min. Subsequently a
selected volume, e.g., about 50 .mu.l, of this mixture is brought
into contact with target cells and the cells are incubated for a
predetermined incubation period, which in the present example was
about 2 hr. The siNA/peptide mixture can optionally include cell
culture medium or other additives such as fetal bovine serum. For
H3, H4 and H2b, a series of experiments was performed to complex
these polynucleotide delivery-enhancing polypeptides with siRNA in
different ratios. Generally this was initiated with a 1:0.01 to
1:50 of siRNA/histone ratio. To each well in a 96-well mircrotiter
plate, 40 .mu.m siRNA was added. Each well contained beta-gal cells
at 50% confluency. Exemplary optimized ratios for transfection
efficiency are shown in Table 2 below.
[0133] Transfections were performed with either regular siRNA or
siRNA complexed with one of the above-identified histone proteins
on 9L/beta-gal cells. The siRNA was designed to specifically knock
down beta-galactosidase mRNA, and activities are expressed as
percentage of beta-gal activities from control (control cells were
transfected using lipofectamine without the polynucleotide
delivery-enhancing polypeptide).
[0134] Assays for detecting and/or quantifying the efficiency of
siRNA delivery are carried out using conventional methods, for
example beta-galactosidase assay or flow cytometry methods.
[0135] For beta-galactosidase assays, 9L/LacZ cells, a cell line
constitutively expressing beta-galactosidase, were used, and the
siRNA against beta-gal mRNA was chemically synthesized and used
with delivery reagents to evaluate knock-down efficiency.
Transfection Procedure
[0136] On the first day of the procedure, saturated 9L/LacZ
cultures are taken from T75 flasks, and the cells are detached and
diluted into 10 ml of complete medium (DMEM, 1.times. PS, 1.times.
Na Pyruvate, 1.times. NEAA). The cells are further diluted to 1:
15, and 100 .mu.l of this preparation are aliquoted into wells of
96 well plates, which will generally yield about 50% cell
confluence by the next day for the transfection. Edges of the wells
are left empty and filled with 250 .mu.l water, and the plates are
placed un-stacked in the incubator overnight at 37.degree. C. (5%
CO.sub.2 incubator).
[0137] On the second day, the transfection complex is prepared in
Opti-MEM, 50 .mu.l each well. The medium is removed from the
plates, and the wells are washed once with 200 .mu.l PBS or
Opti-MEM. The plates are blotted and dried completely with tissue
by invertion. The transfection mixture is then added (50
.mu.l/well) into each well, and 250 .mu.l water is added to the
wells on the edge to prevent them from drying. The cells are then
incubated for at least 3 hours at 37.degree. C. (5% CO.sub.2
incubator). The transfection mixture is removed and replaced with
100 .mu.l of complete medium (DMEM, 1.times. PS, 1.times. Na
-Pyruvate, 1.times. NEAA). The cells are cultured for a defined
length of time, and then harvested for the enzyme assay.
Enzymatic Assay
[0138] Reagents for the enzymatic assay were purchased from
Invitrogen (.beta.-Gal Assay Kit, Catalog no.), and Fisher (Pierce
Micro BCA Protein Assay Reagent Kit, Catalog).
A: Cell Lysis
[0139] Remove the medium, wash once with 200 .mu.l PBS, blot the
plate dry with inversion.
[0140] Add 30 .mu.l lysis buffer from .beta.-Gal Kit into each
well.
[0141] Freeze-Thaw the cells twice to generate lysate.
B: .beta.-Gal Assay
[0142] Prepare assay mix (50 .mu.l 1.times. buffer, 17 .mu.l ONPG
each well)
[0143] Take new plate, add 65 .mu.l assay mix into each well.
[0144] Add 10 .mu.l of cell lysate into each well. There should be
blank wells for subtraction of the background activities.
[0145] Incubate at 37.degree. C. for about 20 minutes, prevent long
incubation which will use up ONPG and biase the high
expression.
[0146] Add 100 .mu.l of the Stop solution.
[0147] Measure the OD at 420 nm.
C: BCA Assay
[0148] Prepare BSA standard (150 .mu.l per well), points should be
duplicated on each plate.
[0149] Put 145 .mu.l of water into each well, add 5 .mu.l of cell
lyaste into each well.
[0150] Prepare final Assay Reagent according to manufacture's
instruction.
[0151] Add 150 .mu.l of Assay Reagent into each well.
[0152] Incubate at 37.degree. C. for about 20 minutes.
[0153] Measure the OD at 562 nm.
D: Calculation of Specific Activity
[0154] The specific activity is expressed as nmol of ONPG
hydrolyzed/t/mg protein, where t is the time of incubation in
minutes at 37.degree. C.; mg protein is the protein assayed which
is determined by BCA method.
Flow Cytometry Measurement of FITC/FAM Conjugated siRNA
[0155] a) After exposure to the complex of siRNA/peptide, cells
were incubated for at least 3 hours.
[0156] b) Wash cells with 200 .mu.l PBS.
[0157] c) Detach cells with 15 .mu.l TE, incubate at 37.degree.
C.
[0158] d) Resuspend cells in five wells with 30 .mu.l FACS solution
(PBS with 0.5% BSA, and 0.1% sodium Azide).
[0159] e) Combine all five wells into a tube.
[0160] f) Add PI (Propidium iodide) 5 .mu.l into each tube.
[0161] g) Analyze the cells with fluorescence activated cell
sorting (FCAS) according to manufacturer's instructions.
[0162] The siRNA sequence used to silence the beta-galactosidase
mRNA was the following: TABLE-US-00002 (SEQ ID NO:)
C.U.A.C.A.C.A.A.A.U.C.A.G.C.G.A.U.U.U.dT.dT (Sense) (SEQ ID NO:)
A.A.A.U.C.G.C.U.G.A.U.U.U.G.U.G.U.A.G.dT.dT (Antisense)
[0163] TABLE-US-00003 TABLE 2 Efficiency of siRNA delivery mediated
by polynucleotide delivery-enhancing polypeptides Delivery
efficiency (% Molar ratio: Peptides of total cells) (siRNA:peptide)
siRNA (40 pmol/well) 0.09 Cationic lipids (Invitrogen) 84.32
unknown Histone H2B 62.03 1:10-15 Histone H3 85.08 1:10-20 Histone
H4 72.07 1:4-8 GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO:) 50.86 1:5-20
WWETWKPFQCRICMRNFSTRQARRNHRRRHR 98.29 1:0.5-4 (SEQ ID NO:) Poly
Lys-Trp, 4:1, MW 20,000-50,000 71.92 1:2-8 Poly Orn-Trp, 4:1, MW
20,000-50,000 74.16 1:2-8
siRNA/Peptide/Lipids
[0164] To evaluate the effects of adding a cationic lipid to a
siNA/polynucleotide delivery-enhancing polypeptide mixture, complex
or conjugate, the above procedures were followed except the
lipofectamine (Invitrogen) was added to siNA/polynucleotide
delivery formulation in constant concentrations, following
manufacturer's instructions (0.2 .mu.l/100 .mu.l Opti-MEM).
[0165] To produce the composition comprised of GKINLKALAALAKKIL
(SEQ ID NO:), siRNA and LIPOFECTIN.RTM. (Invitrogen), the siRNA and
peptide were mixed together first in Opti-MEM cell culture medium
at room temperature, after which LIPOFECTIN.RTM. was added at room
temperature to the mixture to form the siRNA/peptide/cationic lipid
composition.
[0166] To produce the composition comprised of RVIRVWFQNKRCKDKK
(SEQ ID NO:), siRNA and LIPOFECTIN.RTM., the peptide and the
LIPOFECTIN.RTM. were mixed together first in Opti-MEM cell culture
medium, into this mixture was added the siRNA to form the
siRNA/peptide/LIPOFECTIN.RTM. composition.
[0167] To produce the siRNA/peptide/cationic lipid composition
using GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ (SEQ ID NO:) or
GEQIAQLIAGYIDIILKKKKSK (SEQ ID NO: 4) it does not matter in which
order the components are added together to produce the
siRNA/peptide/cationic lipid composition.
[0168] To produce the siRNA/mellitin/LIPOFECTIN.RTM., the siRNA and
mellitin were first mixed together in Opti-MEM cell culture medium
and then the LIPOFECTIN.RTM. was added to the mixture.
[0169] To produce the siRNA/histone H1/LIPOFECTIN.RTM. composition,
the histone H1 and LIPOFECTIN.RTM. were first added together in
Opti-MEM cell culture medium thoroughly mixed and then the siRNA
was added, thoroughly and mixed with the histone LIPOFECTIN.RTM.
mixture to form the siRNA/histone H1/LIPOFECTIN.RTM. composition.
TABLE-US-00004 TABLE 3 Efficiency of siRNA delivery mediated by
polynucleotide delivery- enhancing polypeptides with and without
cationic lipid siRNA:Peptide Delivrery efficiency ratio added in
Delivery efficiency with w/o lipids (% of total transfection
Peptides lipids (% of total cells) cells) mixture siRNA only 1.72
0.11 Lipofectamine 83.48 GKINLKALAALAKKIL 89.67 0.26 1:5-20
RVIRVWFQNKRCKDKK 89 0.59 1:1-5
GRKKRRQRRRPPQGRKKRRQRRRPPQGRKKRRQRRRPPQ 89.99 54.58 1:5
GEQIAQLIAGYIDIILKKKKSK 90.01 50.86 1:5-10 Mellitin 93.1 5.15 1:20
Histone H1 93.39 0.14 1:10-20
[0170] Based on the foregoing results, it is apparent that
exemplary polynucleotide delivery-enhancing polypeptides of the
invention can substantially induce or enhance cellular uptake of
siNAs, while the addition of an optional cationic lipid to certain
siNA/polypeptide mixtures of the invention may substantially
improve siNA delivery efficiency.
EXAMPLE 2
Production and Characterization of Compositions Comprising a siRNA
Comjugated With a TAT-HA Polynucleotide Delivery-Enhancing
Polypeptide
[0171] The present example describes the synthesis and uptake
activity of specific peptides covalently conjugated to one strand
of a siRNA duplex. These conjugates efficiently deliver siRNA into
the cytoplasm and mediate knockdown of desired target genes.
Peptide Synthesis
[0172] Peptides were synthesized by solid-phase Fmoc chemistry on
CLEAR-amide resin using a Rainin Symphony synthesizer. Coupling
steps were performed using 5 equivalents of HCTU and Fmoc amino
acid with an excess of N-methylmorpholine for 40 minutes. Fmoc
removal was accomplished by treating the peptide resin with 20%
piperidine in DMF for two 10 minutes cycles. Upon completion of the
entire peptide, the Fmoc group was removed with piperidine and
washed extensively with DMF. Maleimido modified peptides were
prepared by coupling 3.0 equivalents of 3-maleimidopropionic acid
and HCTU in the presence of 6 equivalents of N-methylmorpholine to
the N-terminus of the peptide resin. The extent of coupling was
monitored by the Kaiser test. The peptides were cleaved from the
resin by the addition of 10 mL of TFA containing 2.5% water and 2.5
triisopropyl silane followed by gentle agitation at room
temperature for 2 h. The resulting crude peptide was collected by
trituration with ether followed by filtration. The crude product
was dissolved in Millipore water and lyophilized to dryness. The
crude peptide was taken up in 15 mL of water containing 0.05% TFA
and 3 mL acetic acid and loaded onto a Zorbax RX-C8 reversed-phase
(22 mm ID.times.250 mm, 5 .mu.m particle size) through a 5 mL
injection loop at a flow rate of 5 mL/min. The purification was
accomplished by running a linear AB gradient of 0.1% B/min where
solvent A is 0.05% TFA in water and solvent B is 0.05% TFA in
acetonitrile. The purified peptides were analyzed by HPLC and
ESMS.
Synthesis of Conjugates
[0173] Both peptides and RNA are prepared using standard solid
phase synthesis methods. The peptide and RNA molecules must be
functionalized with specific moieties to allow for covalent
attachment to each other. For the peptide, the N-terminus is
functionalized, for example, with 3-maleimidopropionic acid.
However, it is recognized that other functional groups such as
bromo or iodoacetyl moieties will work as well. For the RNA
molecule the 5' end of the sense strand or 3' end of the antisense
strand is functionalized with, for example, a
1-O-dimethoxytrityl-hexyl-disulfide linker according to the
following synthetic method.
[0174] The 5' modified C6SS-oligonucleotide
(GCAAGCUGACCCUGAAGUUCAU; 3.467 mg; 0.4582 .mu.mol) was reduced to
the free thiol group with 0.393 mg (3 eq) of
tris(2-carboxyethyl)phosphine (TCEP) in 0.3 ml of 0.1 M
triethylamine acetate (TEAA) buffer (pH 7.0) at room temperature
for 3 h. The reduced oligonucleotide was purified by RP HPLC on
XTerra.RTM.MS C.sub.18 4.6.times.50 mm column using a linear
gradient from 0-30% of CH.sub.3CN in 0.1 M TEAA buffer pH 7 within
20 min (t.sub.r=5.931 min).
[0175] Purified reduced oligonucleotide (1.361 mg, 0.19085 .mu.mol)
was dissolved in 0.2 ml of 0.1 M TEAA buffer pH=7 and then the
peptide with the maleimido moiety attached to the peptide
N-terminus (0.79 mg, 1.5 eq) was added to the oligonucleotide
solution. After addition of peptide a precipitate immediately
formed which disappeared upon the addition of 150 .mu.l of 75%
CH.sub.3CN/0.1M TEAA. After stirring overnight at room temperature,
the resulting conjugate was purified by RP HPLC on XTerra.RTM.MS
C.sub.18 4.6.times.50 mm column using a linear gradient from 0-30%
of CH.sub.3CN in 0.1M TEAA buffer pH 7 within 20 min and 100% C
within next 5 min (t.sub.r=21.007 min). The amount of the conjugate
was determined spectrophotometrically based on the calculated molar
absorption coefficient at .delta.=260 nm. MALDI mass spectrometric
analysis showed that the peak observed for the conjugate (10 585.3
amu) matches the calculated mass. Yield: 0.509 mg, 0.04815 .mu.mol,
25.2%.
[0176] The peptide conjugate sense strand and complimentary
antisense strand were annealed in 50 mM potassium acetate, 1 mM
magnesium acetate and 15 mM HEPES pH 7.4 by heating at 90.degree.
C. for 2 min followed by incubation at 37.degree. C. for 1 h. The
formation of the double stranded RNA conjugate was confirmed by non
denaturing (15%) polyacrylamide gel elctrophoresis and staining
with ethidium bromide. Structure of the Peptide-siRNA Conjugate
##STR1## Uptake Experiments
[0177] Cells were plated the day before in 24-well plates so that
they were .about.50-80% confluent at time of transfection. For
complexes, siRNA and peptide were diluted in Opti-MEM.RTM. media
(Invitrogen), then mixed and allowed to complex 5-10 minutes before
adding to cells washed with PBS. Final concentration of siRNA was
500 nM at each peptide concentration (2-50CM). The conjugate, also
diluted in Opti-MEM.RTM. media, was added to cells at final
concentrations ranging from 62.5 nM to 500 nM. At 500 nM
concentration, we also combined with 20% FBS just before adding to
washed cells. Cells were transfected for 3 hours at 37.degree. C.,
5% CO.sub.2. Cells were washed with PBS, treated with trypsin and
then analyzed by flow cytometry. siRNA uptake was measured by
intensity of Cy5 fluorescence and cellular viability assessed by
addition of propidium iodide.
[0178] As shown in FIGS. 1 and 2, higher uptake and greater mean
fluorescence uptake are observed for the conjugate compared to
simply complexing the peptide and RNA. This indicates that in
certain embodiments it will be desirable to conjugate the
polynucleotide delivery-enhancing polypeptide to the siNA
molecule.
EXAMPLE 3
Screening of siRNA/Delivery Peptide Complexes Demonstrates
Efficient Induction of siRNA Uptake in 9L/LacZ Cells by a Diverse
Assemblage of Rationally-Designed Polynucleotide Delivery-Enhancing
Polypeptides
[0179] The present example provides additional evidence that a
broad and diverse assemblage of rationally-designed polynucleotide
delivery-enhancing polypeptides of the invention induce or enhance
siRNA uptake when complexed with siRNAs
[0180] Approximately 10,000 9L/lacZ cells were plated per well in
flat-bottom 96-well plates so that they would be .about.50%
confluent the next day at the time of transfection. FAM-labeled
siRNA and peptides were diluted in Opti-MEM.RTM. media (Invitrogen)
at 2-fold the final concentration. Equal volumes of siRNA and
peptide were mixed and allowed to complex 5-10 minutes at room
temperature and then 50 .mu.L added to cells, previously washed
with PBS. Cells were transfected for 3 hours at 37.degree. C., 5%
CO.sub.2. Cells were washed with PBS, treated with trypsin and then
analyzed by flow cytometry. siRNA uptake was measured by intensity
of FAM fluorescence and cellular viability assessed by addition of
propidium iodide. The results of these screening assays are
illustrated in Table 4 below. TABLE-US-00005 TABLE 4 Efficiency of
siRNA delivery mediated by rationally-designed polynucleotide
delivery-enhancing polypeptides % Uptake Peptide siRNA (%PI- PN #
Sequence Conc. Conc. /FAM+) PN173 GRKKRRQRRRPPQC (SEQ ID NO:) 10
.mu.M 400 nM 84.8 PN227 Maleimide-AAVALLPAVLLALLA (SEQ ID NO:)
PRKKRRQRRRPPQ-amide 1 .mu.M 400 nM 31.0 PN27
AAVALLPAVLLALLAPRKKRRQRRRPPQC (SEQ ID NO:) 1 .mu.M 400 nM 82.6
PN275 Maleimide- AAVALLPAVLLALLAPRK (SEQ ID NO:) 4 .mu.M 400 nM
95.3 KRRQRRRPPQ-amide PN28 NH2-RKKRRQRRRPPQCAAVALLPAVLLA (SEQ ID
NO:) 2 .mu.M 400 nM 79.3 LLAP-amide PN69 BrAc-GRKKRRQRRRPQ-amide
(SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN81 BrAc-RRRQRRKRGGDIMGEWGNEIFGAI
(SEQ ID NO:) 8 .mu.M 800 nM 97.9 AGFLG-amide PN250
NH2-RRRQRRKRGGDIMGEWGNEIFGAIA (SEQ ID NO:) 15 .mu.M 800 nM 99.5
GFLG-amide PN204 C(YGRKKRRQRRRG)2 (SEQ ID NO:) 1.4 .mu.M 800 nM
82.5 PN280 Maleimide-GRKKRRQRRRPPQ-amide (SEQ ID NO:) 80 .mu.M 400
nM 7.9 PN350 NH2-KLWKAWPKLWKXLWKP-amide (SEQ ID NO:) 10 .mu.M 400
nM 0.0 PN365 AAVALLPAVLLALLAPRRRRRR-amide (SEQ ID NO:) 10 .mu.M 400
nM 81.4 PN366 RLWRALPRVLRRLLRP-amide (SEQ ID NO:) 10 .mu.M 400 nM
0.0 PN29 NH2-AAVALLPAVLLALLAPSGASGLDKR (SEQ ID NO:) 80 .mu.M 400 nM
86.5 DYV-amide PN235 Maleimide-AAVALLPAVLLALLAPSGA (SEQ ID NO:) 80
.mu.M 400 nM 0.0 SGLDKRDYV-amide PN30 NH2-SGASGLDKRDYVAAVAALLPAVLLA
(SEQ ID NO:) 80 .mu.M 400 nM 0.0 LLAP-amide PN202
NH2-LLETLLKPFQCRICMRNFSTRQARR (SEQ ID NO:) 2 .mu.M 400 nM 70.8
NHRRRHRR-amide PN225 NH2-AAVACRICMRNFSTRQARRNHRRRH (SEQ ID NO:) 2
.mu.M 400 nM 30.9 RR-amide PN236 Maleimide-RQIKIWFQNRRMKWKK- (SEQ
ID NO:) 10 .mu.M 400 nM 37.7 amide PN58 RQIKIWFQNRRMKWKK amide (SEQ
ID NO:) 40 .mu.M 400 nM 75.8 PN251 NH2-RQIKIWFQNRRMKWKKDIMGEWGNE
(SEQ ID NO:) 4 .mu.M 400 nM 44.5 IFGAIAGFLG-amide PN279
Maleimide-SGRGKQGGKARAKAKTRSS (SEQ ID NO:) 40 .mu.M 400 nM 24.7
RAGLQFPVGRVHRLLRKG-amide PN61 SGRGKQGGKARAKAKTRSSRAGLQFPVGR (SEQ ID
NO:) 80 .mu.M 800 nM 86.8 VHRLLRKGC-amide PN360
KGSKKAVTKAQKKDGKKRKRSRK- (SEQ ID NO:) 80 .mu.M 400 nM 0.0 amide
PN361 NH2-KKDGKKRKRSRXESYSVYVYKVLKQ (SEQ ID NO:) 10 .mu.M 400 nM
42.0 -amide PN73 KGSKKAVTKAQKKDGKKRKRSRKESYSVY (SEQ ID NO:) 10
.mu.M 400 nM 99.5 VYKVLKQ PN64 BrAc-GWTLNSAGYLLGKINLKALAALAK (SEQ
ID NO:) 10 .mu.M 400 nM 14.5 KILamide PN159 KLALKLALKALKAALKLAamide
(SEQ ID NO:) .08 .mu.M 80 nM 16.4 PN68 BrAc-KLALKLALKALKAALKLAamide
(SEQ ID NO:) 10 .mu.M 400 nM 0.0 PN182 Ac-KETWWETWWTEWSQPKKKRKV-
(SEQ ID NO:) 1 .mu.M 400 nM 84.9 amide PN183
NH2-KETWWETWWTEWSQPGRKKRRQRR (SEQ ID NO:) 20 .mu.M 400 nM 78.1
RPPQ-amide PN71 BrAc-RRRRRRR (SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN87
QqQqQqQqQq (SEQ ID NO:) 10 .mu.M 400 nM 0.0 PN249
NH2-RRRQRRKRGGqQqQqQqQqQ- (SEQ ID NO:) 80 .mu.M 400 nM 0.0 amide
PN158 RVIRWFQNKRCKDKK-amide (SEQ ID NO:) 1 .mu.M 400 nM 94.0 PN86
Ac-LGLLLRHLRHHSNLLANI-amide (SEQ ID NO:) 80 .mu.M 400 nM 62.2 PN162
GQMSEIEAKVRTVKLARS-amide (SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN228
NH2-KLWSAWPSLWSSLWKP-amide (SEQ ID NO:) 80 .mu.M 400 nM 6.8 PN357
NH2-KKKKKKKKK-amide (SEQ ID NO:) 10 .mu.M 400 nM 0.0 PN358
NH2-AARLHRFKNKGKDSTEMRRRR- (SEQ ID NO:) 40 .mu.M 400 nM 0.0 amide
PN283 Maleimide-GLGSLLKKAGKKLKQPKS (SEQ ID NO:) 40 .mu.M 400 nM
36.3 KRKV-amide PN284 Maleimide-Dmt-r-FK-amide (SEQ ID NO:) 100
.mu.M 400 nM 0.0 PN285 Maleimide-Dmt-r-FKQqQqQqQqQq- (SEQ ID NO:) 8
.mu.M 800 nM 90.7 amide PN286 Maleimide-WRFK-amide (SEQ ID NO:) 80
.mu.M 400 nM 0.0 PN289 Maleimide-WRFKQqQ + qQqQqQq- (SEQ ID NO:) 8
.mu.M 400 nM 91.7 amide PN267 Maleimide-YRFK-amide (SEQ ID NO:) 80
.mu.M 400 nM 0.3 PN282 Maleimide-YRFKYRFKYRFK-amide (SEQ ID NO:) 40
.mu.M 800 nM 22.8 PN286 Maleimide-WRFK-amide (SEQ ID NO:) 80 .mu.M
400 nM 0.0 PN290 Maleimide-WRFKKSKRKV-amide (SEQ ID NO:) 80 .mu.M
400 nM 5.3 PN291 Maleimide-WRFKAAVALLPAVLLALLA (SEQ ID NO:) 4 .mu.M
800 nM 12.5 P-amide PN243 NH2-DiMeYrFKamide (SEQ ID NO:) 40 .mu.M
400 nM 0.0 PN244 NH2-YrFKamide (SEQ ID NO:) 80 .mu.M 400 nM 0.0
PN245 NH2-DiMeYRFKamide (SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN246
NH2-WrFKamide (SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN247
NH2-DiMeYrWKamide (SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN248
NH2-KFrDiMeY-amide (SEQ ID NO:) 80 .mu.M 400 nM 0.0 PN287
Maleimide-WRFKWRFK-amide (SEQ ID NO:) 10 .mu.M 400 nM 8.8 PN288
Maleimide-WRFKWRIFKWRFK-amide (SEQ ID NO:) 4 .mu.M 400 nM 9.0
EXAMPLE 4
siRNA/Delivery is Enhanced by Polynucleotide Delivery-Enhancing
Polypeptides in Murine Cells
[0181] The present example illustrates induction/enhancement of
siRNA uptake by polynucleotide delivery-enhancing polypeptides of
the invention in LacZ cells and also in murine primary fibroblasts.
The materials and methods used for these experiments are generally
the same as described above, except that for the murine experiments
9L/LacZ cells were replaced with mouse tail fibroblasts. The
results of these studies are provided in Tables 5 and 6 below.
TABLE-US-00006 TABLE 5 Efficiency of siRNA delivery mediated by
rationally-designed polynucleotide delivery-enhancing polypeptides
in murine fibroblasts % Name Sequence Status Uptake siRNA PN250
NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFLG-amide (SEQ ID NO:) 0.5 .mu.M
siRNA/ 85.9 Cy5- 40 .mu.M peptide eGFP PN73
NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVL (SEQ ID NO:) 0.5 .mu.M
siRNA/ 94.5 Cy5- KQ-amide 5 .mu.M peptide eGFP PEG- Peg- PN509
KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ- (SEQ ID NO:) 0.5 .mu.M sIRNA/
91 Cy5- amide 25 .mu.M peptide eGFP PN404
NH2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVL (SEQ ID NO:) 0.5 .mu.M
siRNA/ 50.4 CyS- RQ-amide 25 .mu.M peptide eGFP PN361
NH2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO:) 0.5 .mu.M siRNA/
65 Cy5- 50 .mu.M peptide eGFP PN27 AAVALLPAVLLALLAPRKKRRQRRRPPQC
(SEQ ID NO:) 0.5 .mu.M sIRNA/ 60.7 Cy5- NO:) 5 .mu.M peptide eGFP
PN58 NH2-RQIKIWFQNRRMKWKK-amide (SEQ ID NO:) 1 .mu.M siRNA/ 3.7
Cy5- 20 .mu.M peptide eGFP PN158 NH2-RVIRWFQNKRCKDKK amide (SEQ ID
NO:) 0.5 .mu.M siRNA/ 86.2 Cy5- 50 nM peptide eGFP PN316
Maleimide-RVIRWFQNKRSKDKK-amide (SEQ ID NO:) 0.5 .mu.M siRNA/ 84.8
Cy5- 100 .mu.M peptide eGFP PN289 Maleimide-WRFKQqQqQqQqQq-amide
(SEQ ID NO:) 0.5 .mu.M siRNA/ 7 Cy5- 10 .mu.M peptide eGFP PN28
NH2-RKKRRQRRRPPQCAAVALLPAVLLALLAP-amide (SEQ ID NO:) 1 .mu.M siRNA/
80.5 Cy5- 8.mu.M peptide eGFP PN173 GRKKRRQRRRPPQC (SEQ ID NO:) 0.5
.mu.M siRNA/ 94.8 Cy5- 130 nM peptide eGFP PN159
KLALKLALKALKAALKLA-amide (SEQ ID NO:) 0.5 .mu.M siRNA/ 0 Cy5- 5
.mu.M peptide eGFP PN161 NH2-GWTLNSAGYLLGKINLKALAALAKKIL-amide (SEQ
ID NO:) 0.5 .mu.M siRNA/ 0 Cy5- 10 nM peptide eGFP
[0182] TABLE-US-00007 TABLE 6 Efficiency of siRNA delivery mediated
by rationally-designed polynucleotide delivery-enhancing
polypeptides in LacZ cells and murine fibrob1asts Percent Uptake
IacZ Primary Peptide Sequence Cells MTF Cells PN27
NH2-AAVALLPAVLLALLAPRKKRRQRRRPPQ-amide (SEQ ID NO:) 86 61 PN28
NH2-RKKRRQRRRPPQAAVALLPAVLLALLAP-amide (SEQ ID NO:) 79 81 PN29
NH2-AAVALLPAVLLALLAPSGASGLDKRDYV-amide (SEQ ID NO:) 87 not tested
PN58 NH2-RQIKIWFQNRRMKWKK-amide (SEQ ID NO:) 76 6 PN61
NH2-SGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLL (SEQ ID NO:) 87 not tested
RKGC-amide PN73 NH2-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVL (SEQ ID NO:)
91 95 KQ-amide PN158 NH2-RVIRWFQNKRCKDKK-amide (SEQ ID NO:) 94 86
PN173 NH2-GRKKRRQRRRPPQC-amide (SEQ ID NO:) 85 95 PN182
NH2-KETWWETWWTEWSQPKKKRKV-amide (SEQ ID NO:) 85 not tested PN202
NH2-LLETLLKPFQCRICMRNFSTRQARRNHRRRH (SEQ ID NO:) 71 not tested
RR-amide PN204 NH2-C(YGRKKRRQRRRG)2-amide (SEQ ID NO:) 83 not
tested PN250 NH2-RRRQRRKRGGDIMGEWGNEIFGAIAGFL (SEQ ID NO:) 99 86
G-amide PN361 NH2-KKDGKKRKRSRKESYSVYVYKVLKQ-amide (SEQ ID NO:) 42
65 PN365 NH2-AAVALLPAVLLALLAPRRRRRR-amide (SEQ ID NO:) 81 not
tested PN404 NH2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYR (SEQ ID NO:) not
50 VLRQ-amide tested PN453 NH2-GALFLGFLGAAGSTMGAWSQPKSKRKVC-amide
(SEQ ID NO:) not 79 tested PN509
Peg-KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKV (SEQ ID NO:) not 91 LKQ-amide
tested
EXAMPLE 5
siRNA/Delivery is Enhanced by Conjugation of the siRNA to
Polynucleotide Delivery-Enhancing Polypeptides
[0183] The present example provides results from screens to
evaluate activity of siRNA/polynucleotide delivery-enhancing
polypeptide conjugates for inducing or enhancing siRNA uptake in
9L/LacZ culture cell lines and primary fibroblast from mouse tail.
The materials and methods employed for these studies are generally
the same as described above, except that no siRNA/peptide mixing is
required as needed to produce siRNA/peptide complexes. The results
of these studies are provided in Tables 7 and 8 below.
TABLE-US-00008 TABLE 7 Efficiency of siRNA delivery mediated by
rationally-designed polynucleotide delivery-enhancing polypeptides
conjugated to siRNAs in LacZ cells Uptake Conjugates Peptide siRNA
% CoP267nfR137-1 YRFK FAM-.beta.-gal tested up 0 to 2.0 uM
CoP286nfR138-1 WRFK FAM-.beta.-gal 0.8 uM 0 CoP287nfR138-1 (WRFK)2
FAM-.beta.-gal 0.8 uM 0 CoP284nfR164-1 Dmt-r-FK FAM-.beta.-gal
tested up 0 to 1.0 uM CoP282nfR165-1 (YRFK)3 FAM-.beta.-gal tested
up 0 to 1.0 uM CoP290nfR165-1 WRFKKSKRKV FAM-.beta.-gal tested up 0
to 1.0 uM CoP277nfR167-1 PN73 FAM-.beta.-gal 1.0 uM 42.9
CoP277nfR167-2 PN73 FAM-.beta.-gal 2.0 uM 55.4
[0184] TABLE-US-00009 TABLE 8 Efficiency of siRNA delivery mediated
by rationally-designed polynucleotide delivery- enhancing
polypeptides conjugated to siRNAs in murine fibroblasts b % Name
Sequence siRNA Status Uptake Cy5- Maleimide-RRRQRRKRGGD Cy5- 0.5
.mu.M 96.3 dsCoP278- IMGEWGNEIFGAIAGFLG- eGFP nfR27O amide dsCoP27-
7 Maleimide- KGSKKAVTKA Cy5- 4 .mu.M 83.5 nfR317
QKKDGKKRKRSRKESYSVYVYK eGFP VLKQ-amide dsCoP275- Maleimide-
AAVALLPAVL Cy5- 4 .mu.M 52.1 nfR321 LALLAPRKKRRQRRRPPQ- eGFP amide
dsCoP285- Maleimide-Dmt-r-FKQ Cy5- 4.mu.M 41.3 nfR3221
qQqQqQqQq-amide eGFP dsCoP236- Maleimide-RQIKIWFQNRR Cy5- 4 .mu.M
36.3 nfR332 MKWKK-amide eGFP dsCoP317- Maleimido-KETWWETWWTE Cy5- 2
.mu.M 29.6 nfR320 WSQPKKKRKV-amide eGFP dsCoP316-
Maleimido-RVIRWFQNKRS Cy5- 2 .mu.M 17.1 nfR347 KDKK-amide eGFP
dsCoP289- Maleimide-WRFKQqQqQqQq Cy5- 4 .mu.M 3.2 nfR268 Qq-amide
eGFP dsCoP276- Maleimide- RKKRRQRRRPP Cy5- 2 .mu.M 3.6 nfR319
QCAAVALLPAVLLALLAP- eGFP amide dsCoP298- NH2-WRFKC-amide Cy5- 4
.mu.M 4.1 cfR248 eGFP dsCoP280- Maleimide-GRKKRRQRRRPPQ- Cy5- 4
.mu.M 1.8 nfR362-1 amide eGFP dsCoP458- Maleimido-KLALKLALKALKAA
CyS- 4 .mu.m 10.8 nfR363-1 LKLA-amide eGFP dsCoP459
Maleimido-GWTLNSAGYLLGKI CyS- 4 .mu.M 54.5 nfR364-1
NLKALAALAKKIL-amide eGFP
[0185] The foregoing data evince that a diverse assemblage of
siRNA/peptide conjugates of the invention mediate delivery of
siRNAs into different cell types at high efficiency.
EXAMPLE 6
siRNA Gene Expression Knock Down is Enhanced by Polynucleotide
Delivery-Enhancing Polypeptides Conjugated to siRNA
[0186] The instant example demonstrates effective knockdown of
target gene expression by siRNA/polynucleotide delivery-enhancing
polypeptide complexes of the invention. In the current studies, the
ability of peptide/siRNA complex to modulate expression of a human
tumor necrosis factor-.alpha. (hTNF-.alpha.) gene, implicated as
mediating the occurrence or progression of RA when overexpressed in
human and other mammalian subjects, was tested.
[0187] Healthy human blood was purchased from Golden West
Biologicals (CA), the peripheral blood mononuclear cells (PBMC)
were purified from the blood using Ficoll-Pague plus (Amersham)
gradient. Human monocytes were then purified from the PBMCs
fraction using magnetic microbeads from Miltenyi Biotech. Isolated
human monocytes were resuspended in IMDM supplemented with 4 mM
glutamine, 10% FBS, 1.times. non-essential amino acid and 1.times.
pen-strep, and stored at 4 C until use.
[0188] In a 96 well flat bottom plate, human monocytes were seeded
at 100K/well/100 .mu.l in OptiMEM medium (Invitrogen). Transfection
reagent was mixed with siRNA at desired concentration in OptiMEM
medium at room temperature for 20 min (for Lipofectamine 200;
Invitrogen), or 5 min (for peptide). At the end of incubation, FBS
was added to the mixture (final 3%), and 50 .mu.l of the mixture
was added to the cells. The cells were incubated at 37 C for 3
hours. After transfection, cells were transferred to V-bottom
plate, and the cells were pelleted at 1500 rpm/5 min. The cells
were resuspended in growth medium (IMDM with glutamine,
non-essential amino acid, and pen-strep).
[0189] After overnight incubation, the cells were stimulated with
LPS (Sigma) at 1 ng/ml for 3 hours. After induction, cells were
collected as above for mRNA quantitation, and supernatant was saved
for protein quantitation.
[0190] For mRNA measurement, branch DNA technology from Genospectra
(CA) was used according to manufacturer's specification. To
quantitate mRNA level in the cells, both house keeping gene (cypB)
and target gene (TNF-.alpha.) mRNA were measured, and the reading
for TNF-.alpha. was normalized with cypB to obtain relative
luminescence unit. To quantify protein level, the TNF-.alpha. ELISA
from BD Bioscience was used according to manufacturer's
specification.
[0191] siRNAs for these studies were directed to target different
regions of the TNF-.alpha. mRNA as illustrated in Table 9 below.
TABLE-US-00010 TABLE 9 Nomenclature and target sequence for siRNAs
targeting TNF-a Alternate SEQ Name Name position Target sequence ID
NO: (SEQ ID NO:) N125 TNF-a-1 516-534 GCGTGGAGCTGAGAGATAA N115
TNF-a-2 430-448 GCCTGTAGCCCATGTTGTA N132 TNF-a-3 738-756
GGTATGAGCCCATCTATCT N108 TNF-a-4 360-378 CCAGGGACCTCTCTCTAAT N138
TNF-a-5 811-829 GCCCGACTATCTCGACTTT N113 TNF-a-6 424-442
TGACAAGCCTGTAGCCCAT N143 TNF-a-7 844-862 GGTCTACTTTGGGATCATT N107
TNF-a-8 359-377 CCCAGGGACCTCTCTCTAA N137 LC1 806-828
AATCGGCCCGACTATCTCGACTT N122 LC2 514-532 AAUGGCGUGGAGCUGAGAGAU N130
LC3 673-691 AACCUCCUCUCUGCCAUCAAG N101 LC4 177-195
AACUGAAAGCAUGAUCCGGGA N140 LC5 820-838 AAUCUCGACUUUGCCGAGUCU N135
LC6 781-799 AAGGGUGACCGACUCAGCGCU N128 LC7 636-654
AAUCAGCCGCAUCGCCGUCUC N127 LC8 612-630 AACCCAUGUGCUCCUCACCCA N118
LC9 472-490 AAGCUCCAGUGGCUGAACCGC N111 LC10 398-416
AAGUCAGAUCAUCUUCUCGAA N110 LC11 363-381 AAGGGACCUCUCUCUAAUCAG N105
LC12 265-287 CCTCAGCCTCTTCTCCTTCCTGA N104 LC13 264-282
AAUCCUCAGCCUCUUCUCCUU N120 LC14 495-513 AACCAAUGCCCUCCUGGCCAA N153
LC16 1535-1555 CTGATTAAGTTGTCTAAACAA N136 LC17 787-807
CCGACTCAGCGCTGAGATCAA N152 LC18 1327-1347 CTTGTGATTATTTATTATTTA
N114 LC19 428-448 AAGCCTGTAGCCCATGTTGTA N145 LC20 982-1002
TAGGGTCGGAACCCAAGCTTA N101 YC-1 177-195 CUGAAAGCAUGAUCCGGGA N103
YC-2 251-269 AGGCGGUGCUUGUUCCUCA N106 YC-3 300-318
CCACCACGCUCUUCUGCCU N109 YC-4 362-380 AGGGACCUCUCUCUAAUCA N113 YC-5
424-442 UGACAAGCCUGUAGCCCAU N115 YC-6 430-448 GCCUGUAGCCCAUGUUGUA
N117 YC-7 435-453 UAGCCCAUGUUGUAGCAAA N120 YC-8 495-513
CCAAUGCCCUCCUGGCCAA N121 YC-9 510-528 CCAAUGGCGUGGAGCUGAG N123
YC-10 515-533 GGCGUGGAGCUGAGAGAUA N125 YC-11 516-534
GCGUGGAGCUGAGAGAUAA N126 YC-12 558-576 GCCUGUACCUCAUCUACUC N130
YC-13 673-691 CCUCCUCUCUGCCAUCAAG N132 YC-14 738-756
GGUAUGAGCCCAUCUAUCU N133 YC-15 772-790 GCUGGAGAAGGGUGACCGA N134
YC-16 776-794 GAGAAGGGUGACCGACUCA N136 YC-17 787-807
GCCCGACUAUCUCGACUUU N141 YC-18 841-859 GCAGGUCUACUUUGGGAUC N143
YC-19 844-862 GGUCUACUUUGGGAUCAUU N144 YC-20 853-871
UGGGAUCAUUGCCCUGUGA N146 YC-21 985-1003 GGTCGGAACCCAAGCTTAG N147
YC-22 1179-1197 CCAGAATGCTGCAGGACTT N148 YC-23 1198-1216
GAGAAGACCTCACCTAGAA N149 YC-24 1200-1218 GAAGACCTCACCTAGAAAT N150
YC-25 1250-1268 CCAGATGTTTCCAGACTTC N151 YC-26 1312-1330
CTATTTATGTTTGCACTTG N154 YC-27 1547-1565 TCTAAACAATGCTGATTTG N155
YC-28 1568-1585 GACCAACTGTCACTCATT
[0192] The foregoing studies demonstrate that siRNAs targeting
TNF-.alpha. expression are effectively delivered in an active state
by polynucleotide delivery-enhancing polypeptides of the invention
to mediate knockdown of TNF-.alpha. expression in monocytes.
TABLE-US-00011 TABLE 10 TNF-.alpha. knockdown mediated by a
PN73/siRNA complex Target Complex Gene siRNA peptide KD (%)
TNF-.alpha. 4 nM 1.6 uM TNF-.alpha. LC1 PN73 20.08 TNF-.alpha. LC2
19.06 TNF-.alpha. LC3 23.17 TNF-.alpha. LC4 26.67 TNF-.alpha. LC5
46.78 TNF-.alpha. LC6 44.10 TNF-.alpha. LC7 42.76 TNF-.alpha. LC8
41.24 TNF-.alpha. LC9 40.32 TNF-.alpha. LC10 13.52 TNF-.alpha. LC11
7.89 TNF-.alpha. LC12 40.61 TNF-.alpha. LC13 48.29 TNF-.alpha. LC14
50.76 TNF-.alpha. LC16 55.91 TNF-.alpha. LC17 50.78 TNF-.alpha.
LC18 63.44 TNF-.alpha. LC19 61.83 TNF-.alpha. LC20 42.68
TNF-.alpha. YC12 43.60
[0193] TABLE-US-00012 TABLE 11 TNF-.alpha. knockdown mediated by a
PN509/siRNA complex Feb. 24, 2005 Target Complex Gene siRNA
peeptide KD (%) TNF-.alpha. 4 nM 1.6 uM TNF-.alpha. LC1 PN509 31.13
TNF-.alpha. LC2 37.04 TNF-.alpha. LC3 30.14 TNF-.alpha. LC4 22.71
TNF-.alpha. LC5 34.93 TNF-.alpha. LC6 50.19 TNF-.alpha. LC7 56.11
TNF-.alpha. LC8 47.35 TNF-.alpha. LC9 58.20 TNF-.alpha. LC10 25.62
TNF-.alpha. LC11 25.65 TNF-.alpha. LC12 17.03 TNF-.alpha. LC13
25.04 TNF-.alpha. LC14 42.78 TNF-.alpha. LC16 40.06 TNF-.alpha.
LC17 48.94 TNF-.alpha. LC18 58.13 TNF-.alpha. LC19 56.38
TNF-.alpha. LC20 71.12 TNF-.alpha. YC12 64.37
[0194] TABLE-US-00013 TABLE 12 TNF-.alpha. knockdown mediated by a
PN250/siRNA complex Feb. 5, 2005 Target Complex Gene siRNA peeptide
KD (%) TNF-.alpha. 20 nM PN250 TNF-.alpha. YC11 0.5 uM 13.70
TNF-.alpha. YC12 17.06 TNF-.alpha. YC17 17.30 TNF-.alpha. YC18
20.72 TNF-.alpha. LC13 20.65 TNF-.alpha. LC20 -3.80 TNF-.alpha.
TNF-4 0.90 TNF-.alpha. YC11 0.75 uM 21.09 TNF-.alpha. YC12 21.66
TNF-.alpha. YC17 29.82 TNF-.alpha. YC18 17.82 TNF-.alpha. LC13
18.04 TNF-.alpha. LC20 10.72 TNF-.alpha. TNF-4 14.39 TNF-.alpha.
YC11 1 uM 33.10 TNF-.alpha. YC12 15.91 TNF-.alpha. YC17 24.68
TNF-.alpha. YC18 24.66 TNF-.alpha. LC13 31.35 TNF-.alpha. LC20
26.53 TNF-.alpha. TNF-4 26.47
The foregoing data evince that effective levels of TNF-.alpha. gene
expression knock down can be achieved in mammalian cells using the
novel siNA/polynucleotide delivery-enhancing polypeptide
compositions of the invention. Screening and Characterization
[0195] FIG. 1 characterizes an exemplary assay system for screening
siRNA candidate sequences for TNF-a knockdown activity. Human
monocytes (CD 14+) treated with LPS induce TNF-.alpha.-specific
mRNA within approximately 2 hrs, followed by peak levels of
TNF-.alpha. protein 2 hrs later. siRNAs were screened for knockdown
activity by transfecting monocytes with siRNA candidate sequences
using Lipofectamine 2000, treating infected cells with LPS, and
measuring TNF-A mRNA levels approximately 16 hrs later. Fifty six
siRNA sequences were designed and screened for their ability to
knockdown TNF-.alpha. mRNA and protein levels in activated human
primary monocytes. Activities for a representative set of 27 siRNA
sequences ranged from 80% mRNA knockdown to no detectable activity.
In general, TNF-.alpha. protein levels were reduced more than mRNA
levels, e.g., a 50% knockdown in TNF-.alpha. mRNA (TNF-.alpha.-1)
resulted in a 75% reduction in TNF-.alpha. protein level. Dose
response curves for selected siRNAs that exhibited knockdown levels
from 30 to 60 % were obtained. Calculated IC.sub.50values were in
the 10-200 pMolar range. While the siRNA sequences evaluated were
distributed throughout the TNF-.alpha. transcript, the most potent
siRNAs identified were located in two areas: the middle of the
coding region and the 3'-UTR.
EXAMPLE 7
siRNA Gene Expression Knock Down is Enhanced by Polynucleotide
Delivery-Enhancing Polypeptides Complexed with siRNA
[0196] The present example demonstrates knockdown of target gene
expression by peptide-siRNA conjugates of the invention. The
materials and methods for these studies are the same as those
described above, with the exception that no mixing of the siRNA and
peptide is required. In the present series of studies, the
knockdown experiments included comparison of siRNA/peptide-mediated
knockdown with and without lipofectamine. TABLE-US-00014 TABLE 13
siRNA/peptide-mediated knockdown of TNF-.alpha. expression with and
without lipofectamine with Lipofectamine without Lipofectamine
peptide siRNA cells in assay conc. (uM) KD (%) conc. (uM) KD (%)
CoP456 cIBR LC20 CD14 0.4 no KD 0.4 no KD 1.3 no KD 1.3 no KD 4 no
KD 4 no KD CoP457 Peptide T LC20 0.4 no KD 0.4 no KD 1.3 no KD 1.3
no KD 4 no KD 4 no KD CoP278 TAT + HA YC12 0.4 no KD 0.4 no KD 1.3
no KD 1.3 no KD 4 no KD 4 no KD CoP277 PN73 LC13 MTF 0.19 31.95
0.19 61.61 0.38 32.83 0.38 76.31 0.75 39.29 0.75 73.94 1.50 41.42
1.50 73.14 3.00 39.88 3.00 58.14 6.00 20.23 6.00 50.71 CoP277 PN73
LC13 CD14 0.000 93.06 0.002 83.63 0.011 72.58 0.053 73.52 0.266
85.01 CoP277 PN73 LC20 CD14 0.000 75.15 0.002 60.72 0.011 57.09
0.053 58.70 0.266 62.79
[0197] The foregoing data evince that a diverse assemblage of
polynucleotide delivery-enhancing polypeptides of the invention
complexed with siRNAs function to enhance siRNA-mediated knockdown
of TNF-gene expression in mammalian subjects.
EXAMPLE 8
Time Course of siRNA Gene Expression Knock Down
[0198] The instant example presents studies relating to the time
course of siRNA-mediated gene expression knockdown. To test the
duration of the siRNA effect, the siRNA transfection procedures as
noted above were employed, except that fibroblasts derived from
eGFP expressing mice were used. The transfection reagent used here
was lipofectamine. The cells were replated on the 18.sup.th day due
to overgrowth. The second trasnfection was performed on the
19.sup.th day post first transfection. On the 19.sup.th day the
eGFP levels were measured after the transfection. Scramble or
nonsense siRNA(Qiagen) was used as a control, along with a GFPI
siRNA (GFPI) and a hairpin siRNA (D#21). The knockdown activities
were calibrated with scramble siRNA (Qiagen control) TABLE-US-00015
TABLE 14 Time Course of siRNA Gene Expression Knock Down Days post
first transfection 1 3 5 7 9 11 13 15 17 19 20 21 25 27 Qiagen 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
GFPI 27.61 60.87 64.75 58.40 56.72 40.46 35.56 16.59 15.50 59.60
37.10 57.38 66.94 59.63 D#21 28.22 61.11 66.91 62.86 57.36 54.71
42.96 24.66 9.88 46.36 35.89 65.25 74.15 58.39
[0199] The foregoing studies demonstrate that siRNA knockdown
activity became apparent around day 3, and was sustained through
day 9, whereafter target gene expression returned to baseline
levels around day 17. After the second transfection on day 18,
another reduction of eGFP expression occurred indicating that the
reagent can be repeatedly administered to cells to yield repeated
or enduring gene expression knockdown.
EXAMPLE 9
Dosage Dependence of TNF Gene Expression Knock Down mediated by
siRNA Complexed with Polynucleotide Delivery-Enhancing
Polypeptide
[0200] The present exemple demonstrates that knockdown activity
mediated by siRNA complexed with an exemplary polynucleotide
delivery-enhancing polypeptide, PN73, in activated human monocytes
is dosage dependent.
[0201] The siRNA/PN73 complex was provided in a constant ratio
between PN73 and siRNA of about PN73:siRNA=82:1. 400 nM siRNA was
complexed with 33 .mu.M PN73 for 5 min in OptiMEM medium. After
complexation, the complex were serial diluted (1:2 ratio) with
OptiMEM. The complex was added to human monocytes for transfection.
The following induction and mRNA quantification was performed
according to the description above. TABLE-US-00016 TABLE 15 Peptide
Dosage Dependence of TNF Gene Expression Knock Down PN73:siRNA
ratio = 82:1 PN73 (uM) siRNA (nM) Control TNF-2 TNF-4 LC8 0 100 100
100 100 1.2 14.81 99.99 80.28 70.22 73.44 3.6 44.44 100.11 69.33
62.97 63.04 11 133.33 99.99 57.82 62.71 59.57 33 400.00 99.99 64.51
78.48 51.30
[0202] In a related series of experiments, siRNA was serially
diluted and combined with a fixed amount of PN73 (1.67 .mu.M).
Alternatively stated, the PN73 polynucleotide delivery-enhancing
polypeptide was complexed with titration amounts of siRNA. PN73
(1.67 uM) was complexed with each titration amount of siRNA for 5
min at RT in OptiMEM medium. After complexation, the complex was
added to human monocytes for transfection. The induction and mRNA
quantification data provided in Table 16, below, were obtained by
methods described above. TABLE-US-00017 TABLE 16 siRNA Dosage
Dependence of TNF-.alpha. Gene Expression Knock Down 1.67 uM PN73
complexed with titration amount of siRNA siRNA conc. (nM) Control
LC20 0.8 100.0 84.7 4 100.0 59.4 20 100.0 65.2 100 100.0 54.7
EXAMPLE 10
Multiple Dosing Protocol to Extend siRNA Knockdown Effect in
Mammalian Cells
[0203] The instant example demonstrates that multiple dosing
schedules will effectively extend gene expression knockdown effects
in mammalian cells mediated by siNA/polynucleotide
delivery-enhancing polypeptide compositions of the invention. The
materials and methods employed for these studies are the same as
described above, with the exception that repeated transfections
were conducted at the times indicated. The scramble siRNA (Qiagen)
was utilized for side by side controls. TABLE-US-00018 TABLE 17
Repeated siRNA TNF-.alpha. Gene Expression Knock Down Days post 1st
transfection 4 5 6 7 8 9 10 11 12 Single 74.69 61.87 62.57 55.47
41.41 39.42 27.21 2nd on 66.69 69.78 68.27 64.18 63.86 64.37 56.52
5th 2.sup.nd on 64.21 65.78 67.74 64.12 58.64 53.96 6th 2.sup.nd on
63.03 62.50 69.94 62.63 58.07 7th
[0204] The foregoing studies demonstrate that when multiple
transfections are performed timely (in this case between about the
5.sup.th-7.sup.th day post first transfection), gene expression
knockdown effects in mammalian cells can be prolonged or
re-induced.
EXAMPLE 11
In Vivo siRNA/Peptide-Mediated TNF-A Gene Expression Knock Down
[0205] The present example provides In Vivo studies demonstrating
the efficacy of siRNA/polynucleotide delivery-enhancing polypeptide
compositions of the invention to mediate systemic delivery and
therapeutic gene knockdown by siRNA, effective to modulate target
gene expression and modify phenotype of cells in a therapeutic
manner.
[0206] Human NF-.alpha. expressing mice were purchase from the
Hellenic Pasture Institute, Greece) at 5 weeks old. Mice were
administered through i.v. with 300 .mu.l saline twice a week (4
mice), with the RA drug Ramicade (5 mg/kg) once a week (2 mice), or
with N145 siRNA (2 mg/kg) mixed with PN73 at 1:5 molar ratio twice
a week (2 mice).
[0207] During the injection periods, plasma samples were collected
for ELISA testing (R&D Systems, Cat#SSTA00C), and paw scores
were taken twice a week as an accepted index of RA disease
progression and therapeutic efficacy. TABLE-US-00019 TABLE 18
hTNF-a ELISA Age(week) 7 8 9 Ramicade 102.24 39.27 25.80 N145/PN73
25.96 21.89 14.21 Saline 33.78 34.29 24.48 *These data represent
the average of the mice in the experiment in pg/ml.
[0208] The foregoing data demonstrate effective reduction of
hTNF-.alpha. levels in siRNA/peptide-treated mice in the
circulating blood as compared to levels in Ramicade or saline
(control) treated mice.
[0209] Additional evidence of in vivo efficacy of the
siNA/polynucleotide delivery-enhancing polypeptide compositions and
methods of the invention were obtained from the above murine
subjects using paw scores, an accepted phenotypic index for RA
disease status and treatment efficacy. Due to the difference in the
starting point (some animals present with scores at earlier
points), the scores have been adjusted to 0 for all animals in the
experiments. Each paw is given a score between 0 and 3, with the
highest score of 12, according to the following scoring index.
[0210] 0: Normal [0211] 1: edema or distortion of paw or ankle
joints [0212] 2: distortion of paw and ankle joints [0213] 3:
ankylosis of wrist or ankle joints.
[0214] The results of these paw score evaluations are presented
graphically in FIG. 3. The data demonstrate that the siRNA/peptide
injected animals showed a delayed RA progression which was
comparable to that exhibited by the Ramicade-treated mice. The
results from the foregoing studies demonstrate that small
interfering nucleic acid and polynucleotide delivery-enhancing
polypeptide compositions of the invention provide promising new
therapeutic tools for regulating gene expression and treating and
managing disease. siNAs of the invention, for example siNAs
targeting human hTNF-.alpha.-specific mRNAs for degradation, offer
higher specificity, lower immunogenicity and greater disease
modification than current small molecule, soluble receptor, or
antibody therapies for RA. More than 50 candidate siRNA sequences
were screened that targeted hTNF-.alpha. and yielded single
administration knockdowns of 30-85%. Over 20 in silico designed
peptide complex and/or covalent molecules were compared for
fluorescent RNA uptake by monocytes and a number were found to have
significantly better uptake than Lipofectamine or
cholesterol-conjugated siRNA and with <10 .mu.M IC.sub.50
values. The peptide-siRNA formulations efficiently knockdown
TNF-.alpha. mRNA and protein levels in activated human monocytes in
vitro.
[0215] One exemplary candidate siRNA/delivery peptide formulation
was evaluated in two transgenic mouse models of rheumatoid
arthritis (RA) constitutively expressing human TNF-.alpha.. Animals
treated with 2 mg/kg siRNA by IV injection or infliximab twice
weekly beginning at age 6 weeks showed RA score stabilization (paw
and joint inflammation) beginning at age 7 weeks, compared to
controls where these disease conditions persisted through week 10.
At age 9 weeks, siRNA treated animals showed comparable reductions
in RA scores, but significantly lower plasma TNF-.alpha. protein
levels than infliximab treated animals.
[0216] Based on the disclosure herein, the use of siRNA to inhibit
the expression of target genes, for example cytokines such as
TNF-.alpha., that play important roles in pathological states, such
as inflammation, provides effective treatments to alleviate or
prevent symptoms of disease, as exemplified by RA, in mammalian
subjects. Exemplary siRNA/peptide compositions employed within the
methods and compositions of the invention provide advantages
relating to their ability to reduce or eliminate target gene
expression, e.g., TNF-.alpha. expression, rather than by complexing
with the product of the target gene, e.g., TNF-.alpha., as in the
case of antibodies or soluble receptors.
[0217] Improving systemic delivery of nucleic acids according to
the teachings of the invention provides yet additional advantages
for development of siNAs as drugs. Specific challenges in this
context include delivery through tissue barriers to a target cell
or tissue, maintaining the stability of the siNA, and intracellular
delivery--getting siNAs across cell membranes into cells in
sufficient quantities to be effective. The present disclosure
demonstrates for the first time an effective in vivo delivery
system comprising novel peptide-siRNA compositions targeting
specific gene expression, such as expression of human TNF-.alpha.,
which attenuate disease activity in transgenic animal models
predictive of target diseases, as exemplified by studies using
murine models of RA. In related studies, the compositions and
methods of the invention effectively inhibit TNF-.alpha. expression
in activated monocytes derived from patients with RA. These results
indicate that the RNAi pathway effectively mediates alteration of
cellular phenotype and disease progression through intracellular
effects on TNF-pathways, and avoids toxicity effects due to
circulating antibody/TNF-.alpha. complexes with residual
immunoreactivity that characterize current antibody therapies for
RA. Notably, all of the tests herein were implemented with
associated toxicity effects minimized, such that the dosages of
siNAs and polynucleotide delivery-enhancing polypeptides shown in
these examples always correlated with cell viability levels of at
least 80-90% or greater.
EXAMPLE 12
Optimizing Rational Design of Polynucleotide Delivery-Enhancing
Polypeptides
[0218] The instant example provides exemplary study design and data
for optimizing rational design of polynucleotide delivery-enhancing
polypeptides of the invention. The subject rational design
manipulations were conducted for a histone H2B-derived
polynucleotide delivery-enhancing polypeptide. TABLE-US-00020 TABLE
19 Deletion and modification of PN73 ##STR2##
[0219] The above Table 19 provides a diagram of the primary
structure of PN73 and its derivatives generated for optimizing
rational design of PN73-based polynucleotide delivery-enhancing
polypeptides. The parent peptide PN73 was demonstrated above to be
an excellent example of polynucleotide delivery-enhancing
polypeptides for inducing or enhancing siRNA delivery to cells. In
order to better understand the function-structural activity
relationships of this and other polynucleotide delivery-enhancing
polypeptides, primary structural studies were performed by
characterizing C- and N-terminal function, and activity of
conjugates between PN73 and other chemical moieties.
[0220] As noted above, PN73 is a peptide from histone 2B, residues
12-48 aa. PN360 is C-terminal deleted version of PN73 (12-35) and
PN361 id N-terminal deleted version of PN73 (23-48). PN404 is a
version of PN73 in which all of lysines are replaced with arginines
as shown below: NH2-RGSRRAVTRAQRRDGRRRRRSRRESYSVYVYRVLRQ-amide (SEQ
ID NO:) PN509 is a pegylated PN73 (PEG molecular weight 1 k Dalton)
derivative that is pegylated at the N-terminus.
[0221] FIG. 4 provides the results of uptake efficacy and viability
studies in mouse fibroblasts for the foregoing PN73
rationally-designed derivative polynucleotide delivery-enhancing
polypeptides. The activity changes of modified PN73 in mouse tail
fibroblast cells are illustrated. Unlike PN404, PN509 increases
uptake without increasing toxicity. While deleting part of the
N-terminus of PN73 reduces activity, removal of C-terminal residues
abolishes the activity. Both PN73 and PN509 show higher activity in
primary cells than Lipofectamine (Invitrogen, Calif.). The uptake
measurements were performed using mouse tail fibroblast cells.
[0222] Although the foregoing invention has been described in
detail by way of example for purposes of clarity of understanding,
it will be apparent to the artisan that certain changes and
modifications may be practiced within the scope of the appended
claims which are presented by way of illustration not limitation.
In this context, various publications and other references have
been cited within the foregoing disclosure for economy of
description. Each of these references is incorporated herein by
reference in its entirety for all purposes. It is noted, however,
that the various publications discussed herein are incorporated
solely for their disclosure prior to the filing date of the present
application, and the inventors reserve the right to antedate such
disclosure by virtue of prior invention.
* * * * *