In vivo gene silencing by chemically modified and stable siRNA
First Claim
1. A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, such that in vivo stability is enhanced as compared to a corresponding unmodified siRNA.
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Abstract
The present invention provides compositions for RNA interference and methods of use thereof. In particular, the invention provides small interfering RNAs (siRNAs) having modification that enhance the stability of the siRNA without a concomitant loss in the ability of the siRNA to participate in RNA interference (RNAi). The invention also provides siRNAs having modification that increase targeting efficiency. Modifications include chemical crosslinking between the two complementary strands of an siRNA and chemical modification of a 3′ terminus of a strand of an siRNA. Preferred modifications are internal modifications, for example, sugar modification, nucleobase modification and/or backbone modifications. Such modifications are also useful, e.g., to improve uptake of the siRNA by a cell. Functional and genomic and proteomic methods are featured. Therapeutic methods are also featured.
488 Citations
83 Claims
- 1. A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, such that in vivo stability is enhanced as compared to a corresponding unmodified siRNA.
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2. A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi) and wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, such that the target efficiency is enhanced compared to a corresponding unmodified siRNA.
- 23. The siRNA of any one of the preceeding claims, wherein the sense strand is crosslinked to the antisense strand
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30. The siRNA of any one of the preceeding claims, wherein a 3′
- OH terminus of the sense strand or antisense strand is modified.
- 53. A method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism the siRNA of any one of the preeceeding claims, said siRNA being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.
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63. A method of treating a disease or disorder associated with the activity of a protein specified by a target mRNA in a subject, comprising administering to said subject the siRNA of any one of the preceeding claims, said siRNA being administered in an amount sufficient for degradation of the target mRNA to occur, thereby treating the disease or disorder associated with the protein.
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64. A method for deriving information about the function of a gene in a cell or organism comprising:
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(a) introducing into said cell or organism the siRNA of any one of the preceeding claims; and
(b) maintaining the cell or organism under conditions such that target-specific RNAi can occur;
(c) determining a characteristic or property of said cell or organism; and
(d) comparing said characteristic or property to a suitable control, the comparison yielding information about the function of the gene.
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65. A method of validating a candidate protein as a suitable target for drug discovery comprising:
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(a) introducing into a cell or organism the siRNA of any one of the preceeding claims; and
(b) maintaining the cell or organism under conditions such that target-specific RNAi can occur;
(c) determining a characteristic or property of said cell or organism; and
(d) comparing said characteristic or property to a suitable control, the comparison yielding information about whether the candidate protein is a suitable target for drug discovery.
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66. A kit comprising reagents for activating target-specific RNA interference (RNAi) in a cell or organism, said kit comprising:
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(a) the siRNA molecule of any one of the preceeding claims; and
(b) instructions for use.
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67. A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi), wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, and wherein the antisense strand is capable of adopting an A-form helix when in association with a target RNA.
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68. A small interfering RNA (siRNA), comprising a sense strand and an antisense strand, wherein the antisense strand has a sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi), wherein the sense strand or antisense strand is modified by the substitution of at least one internal nucleotide with a modified nucleotide, and wherein the antisense strand is capable of adopting an A-form helix having a normal major groove when in association with a target RNA.
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69. An siRNA derivative comprising an siRNA having two complementary strands of nucleic acid, wherein the two strands are crosslinked, a 3′
- OH terminus of one of the strands is modified, or the two strands are crosslinked and modified at the 3′
OH terminus. - View Dependent Claims (70, 71)
- OH terminus of one of the strands is modified, or the two strands are crosslinked and modified at the 3′
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72. A method comprising contacting a cell with a concentration of an siRNA derivative sufficient to inhibit expression of a target gene, wherein the siRNA derivative:
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(a) is a crosslinked siRNA;
(b) contains a single crosslink;
(c) is psoralen crosslinked. (d) is modified at a 3′
terminus(e) comprises a biotin at a 3′
terminus.(f) comprises a photocleavable biotin having the structure depicted in FIG. 8 at a 3′
terminus.(g) comprises a peptide, nanoparticle, peptidomimetic, or dendrimer at a 3′
terminus.(h) comprises a Tat peptide at a 3′
terminus. - View Dependent Claims (73, 74, 75, 76, 77, 78)
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79. A photocleavable biotin of the formula depicted in
FIG. 20 .
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80. A method of determining whether a candidate siRNA derivative is an siRNA derivative, the method comprising
(a) obtaining a reporter cell comprising two different fluorescent reporter genes, (b) transfecting the reporter cell with a candidate siRNA derivative targeted to one of the fluorescent reporter genes, thereby creating a test cell; -
(c) incubating the test cell for a time sufficient for a reporter cell to express detectable levels of the fluorescent reporter proteins encoded by the fluorescent reporter genes;
(d) determining the fluorescence intensity of each fluorescent reporter protein in the test cell; and
(e) determining the ratio of the level of fluorescence intensity between the two fluorescent reporter proteins in the test cell and normalizing the ratio to the ratio of fluorescence intensity in a control reporter cell that was not transfected with the candidate siRNA derivative, wherein a normalized ratio of less than one indicates that the candidate siRNA derivative is an siRNA derivative. - View Dependent Claims (81, 82, 83)
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Specification