Extended Dicer Substrate Agents and Methods for the Specific Inhibition of Gene Expression
The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a pattern of deoxyribonucleotides (in most embodiments, the pattern comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair) designed to direct the site of Dicer enzyme cleavage within the dsNA molecule. Deoxyribonucleotides of the dsNA molecules of the invention are located within a region of the dsNA that can be excised via Dicer cleavage to generate an active siRNA agent that no longer contains the deoxyribonucleotide pattern (e.g., deoxyribonucleotide-deoxyribonucleotide base pairs). Such DNA-extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be more effective RNA inhibitory agents than corresponding double stranded RNA-extended DsiRNAs. DsiRNA agents were also found to tolerate guide strand mismatches.
1 - 106 . (canceled)
107 . An isolated double stranded nucleic acid (dsNA) comprising a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein:
said first strand is 31 to 60 nucleotide residues in length,
said second strand is 31 to 60 nucleotide residues in length;
said second strand anneals to said first strand,
said second strand comprises a 3′ overhang of unmodified and modified nucleotides,
said second strand is complementary to a sequence of a target mRNA of a target gene; and
said second strand is sufficiently complementary to a target RNA to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.
108 . The isolated dsNA of claim 107 , wherein the overhang of the second strand is conjugated to a non-nucleic acid moiety.
109 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety is a peptide or an organic compound.
110 . The isolated dsNA of claim 109 , wherein the organic compound is a dye.
111 . The isolated dsNA of claim 110 , wherein the organic compound is cholesterol.
112 . The isolated dsNA of claim 107 , in combination with a sugar
113 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety increases cellular uptake.
114 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety increase cellular targeting of the dsNA.
115 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety increases stability of the dsNA.
116 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety facilitates tracking of the dsNA.
117 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety increases the binding affinity of the dsNA.
118 . The isolated dsNA of claim 108 , wherein the non-nucleic acid moiety decreases the immunogenicity of the dsNA.
119 . The isolated dsNA of claim 107 , wherein the modified nucleotide is a phosphorothioate-modified nucleotide residue (PS-NA).
120 . The isolated dsNA of claim 107 , wherein the modified nucleotide is a peptide nucleic acid (PNA).
121 . The isolated double stranded nucleic acid of claim 107 , wherein the modified nucleotide comprises a modified base and/or a modified sugar moiety.
122 . The isolated double stranded nucleic acid of claim 107 , wherein the modified nucleotide is selected from the group consisting of: dideoxyribonucleotides, acyclonucleotides 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).
123 . The isolated double stranded nucleic acid of claim 121 , wherein the modified sugar moiety is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methylcarbamate).
124 . The isolated double stranded nucleic acid of claim 107 , wherein at least one internucleoside linkage is modified.
125 . The isolated double stranded nucleic acid of claim 124 , wherein the internucleoside linkage modification is selected from the group consisting of:
methylphosphonate, phosphorothioate, and phosphotriester modifications.
126 . The isolated double stranded nucleic acid of claim 107 , further comprising a fluorescein.
127 . The isolated double stranded nucleic acid of claim 107 , wherein the modified nucleotide comprises an LNA modification.
128 . The isolated dsNA of claim 107 , wherein the modified nucleotide is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate).
129 . The isolated dsNA of claim 107 , wherein the modified nucleotide of said 3′ overhang is a 2′-O-methyl ribonucleotide.
130 . The isolated dsNA of claim 107 , wherein all nucleotides of said 3′ overhang are modified nucleotides.
131 . The isolated dsNA of claim 107 , wherein one or both of said first and second strands comprises a 5′ phosphate.
132 . The isolated dsNA of claim 107 , wherein the 3′ overhang is two nucleotides in length and wherein said modified nucleotide of said 3′ overhang is a 2′-O-methyl modified ribonucleotide.
133 . The isolated dsNA of claim 107 , wherein the first strand has a nucleotide sequence that is at least 80%, 90%, 95% or 100% complementary to the second strand nucleotide sequence.
134 . The isolated dsNA of claim 107 , wherein a nucleotide of said second or first oligonucleotide strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.
135 . The isolated dsNA of claim 107 , wherein the 3′ terminus of said first strand and the 5′ terminus of said second strand are joined by a chemical linker.
136 . The isolated dsNA of claim 107 , wherein the dsNA is a dicer substrate.
137 . The isolated dsNA of claim 107 , wherein the dsNA is a Dicer substrate that, upon endogenous Dicer processing, yields double-stranded nucleic acids of 19-23 nucleotides in length capable of reducing target gene expression in a mammalian cell.
138 . The isolated dsNA of claim 107 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.
139 . The isolated dsNA of claim 107 , wherein the dsNA reduces target gene expression in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.
140 . The isolated dsNA of claim 107 , wherein the dsNA, when introduced into a mammalian cell, reduces target gene expression by at least 70% when transfected into said cell at a concentration selected from the group consisting of 1 nM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less and 10 pM or less.
141 . The isolated dsNA of claim 107 , wherein at least 50% of the ribonucleotide residues of said dsNA are unmodified ribonucleotides.
142 . The isolated dsNA of claim 107 , wherein at least 50% of the ribonucleotide residues of said second strand are unmodified ribonucleotides.
143 . The isolated dsNA of claim 107 , wherein the second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5′ terminal nucleotide residue of said first oligonucleotide strand and toward the 5′ end of said second strand, comprises alternating modified and unmodified nucleotide residues.
144 . The isolated dsNA of claim 107 , wherein the first strand is of a length selected from the group consisting of: 33 to 49 nucleotides, 35 to 49 nucleotides and 37 to 49 nucleotides.
145 . The isolated dsNA of claim 107 , wherein the second strand possesses a 3′ overhang of 1-4 nucleotides in length.
146 . The isolated dsNA of claim 145 , wherein the 3′ overhang is 1-3 nucleotides in length.
147 . The isolated dsNA of claim 145 , wherein the 3′ overhang is 1-2 nucleotides in length.
148 . A method for reducing expression of a target gene in a cell, comprising: contacting a cell with an isolated double stranded NA (dsNA) as claimed in claim 107 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.
149 . A method for reducing expression of a target gene in an animal, comprising: treating an animal with an isolated double stranded NA (dsNA) as claimed in claim 107 in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.
150 . The method of claim 149 , wherein the dsNA possesses enhanced pharmacokinetics when compared to an appropriate control DsiRNA.
151 . The method of claim 149 , wherein the dsNA possesses enhanced pharmacodynamics when compared to an appropriate control DsiRNA.
152 . The method of claim 149 , wherein the dsNA possesses reduced toxicity when compared to an appropriate control DsiRNA.
153 . The method of claim 149 , wherein the dsNA possesses enhanced intracellular uptake when compared to an appropriate control DsiRNA.
154 . A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising the isolated double stranded NA (dsNA) of claim 107 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.
155 . A method of synthesizing the double stranded NA (dsNA) of claim 107 , comprising chemically or enzymatically synthesizing said dsNA.
156 . A kit comprising the dsNA of claim 107 and instructions for its use.
157 . An isolated double stranded nucleic acid (dsNA) comprising a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein: the first strand is 31 to 60 nucleotide residues in length, wherein starting from the first nucleotide (position 1) at the 5′ terminus of the first strand, positions 1 to 23 of said first strand are ribonucleotides; said second strand is 31 to 60 nucleotide residues in length and comprises 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of said first strand to form a duplex; the 5′ terminus of said first strand and the 3′ terminus of said second strand form a structure selected from the group consisting of a blunt end and a 1-4 nucleotide 3′ overhang; the 3′ terminus of said first strand and the 5′ terminus of said second strand form a duplexed blunt end; at least one of positions 24 to the 3′ terminal nucleotide residue of said first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand; and said second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of said second strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.
158 . The isolated dsNA of claim 157 , wherein two or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
159 . The isolated dsNA of claim 157 , wherein four or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
160 . The isolated dsNA of claim 157 , wherein six or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
161 . The isolated dsNA of claim 157 , wherein eight or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
162 . The isolated dsNA of claim 157 , wherein ten or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
163 . The isolated dsNA of claim 157 , wherein twelve or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
164 . The isolated dsNA of claim 157 , wherein fourteen or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
165 . The isolated dsNA of claim 157 , wherein sixteen or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
166 . The isolated dsNA of claim 157 , wherein eighteen or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
167 . The isolated dsNA of claim 157 , wherein twenty or more nucleotide residues of positions 24 to the 3′ terminal nucleotide residue of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
168 . The isolated dsNA of claim 158 , wherein the deoxyribonucleotides of said first strand that base pair with said deoxyribonucleotides of said second strand are consecutive deoxyribonucleotides.
169 . The isolated dsNA of claim 157 , wherein two or more consecutive nucleotide residues of positions 24 to 27 of said first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of said second strand.
170 . The isolated dsNA of claim 157 , wherein each of positions 24 and 25 of said first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand.
171 . The isolated dsNA of claim 157 , wherein each nucleotide residue of positions 24 to 27 of said first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand.
172 . The isolated dsNA of claim 157 , wherein each nucleotide residue of positions 24 to 29 of said first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand.
173 . The isolated dsNA of claim 157 , wherein each nucleotide residue of positions 24 to 31 of said first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand.
174 . The isolated dsNA of claim 157 , wherein positions 24 to the 3′ terminal nucleotide residue of said first strand comprise between one and 25 deoxyribonucleotide residues, wherein each of said deoxyribonucleotide residues of said first strand base pairs with a deoxyribonucleotide of said second strand.
175 . The isolated dsNA of claim 157 , wherein the deoxyribonucleotides of said second strand that base pair with said deoxyribonucleotides of said first strand are not complementary to said target RNA.
176 . The isolated dsNA of claim 157 , wherein the second strand possesses a 3′ overhang of 1-4 nucleotides in length.
177 . The isolated dsNA of claim 176 , wherein the 3′ overhang is 1-3 nucleotides in length.
178 . The isolated dsNA of claim 176 , wherein the 3′ overhang is 1-2 nucleotides in length.
179 . The isolated dsNA of claim 176 , wherein the nucleotides of said 3′ overhang comprise a modified nucleotide.
180 . The isolated dsNA of claim 179 , wherein the modified nucleotide residue is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate).
181 . The isolated dsNA of claim 179 , wherein the modified nucleotide of said 3′ overhang is a 2′-O-methyl ribonucleotide.
182 . The isolated dsNA of claim 179 , wherein all nucleotides of said 3′ overhang are modified nucleotides.
183 . The isolated dsNA of claim 157 , wherein one or both of said first and second strands comprises a 5′ phosphate.
184 . The isolated dsNA of claim 179 , wherein the 3′ overhang is two nucleotides in length and wherein said modified nucleotide of said 3′ overhang is a 2′-O-methyl modified ribonucleotide.
185 . The isolated dsNA of claim 176 , wherein the second strand, starting from the nucleotide residue of said second strand that is complementary to the 5′ terminal nucleotide residue of said first oligonucleotide strand (position 1*), comprises unmodified nucleotide residues at all positions from position 20* to the 5′ terminal residue of said second strand.
186 . The isolated dsNA of claim 157 , wherein starting from the first nucleotide (position 1*) at the 3′ terminus of said first strand, position 1*, 2* and/or 3* is a deoxyribonucleotide.
187 . The isolated dsNA of claim 186 , wherein the first strand comprises a deoxyribonucleotide at position 1* from the 3′ terminus of said first strand.
188 . The isolated dsNA of claim 186 , wherein the first strand comprises deoxyribonucleotides at positions 1* and 2* from the 3′ terminus of said first strand.
189 . The isolated dsNA of claim 157 , wherein the ultimate and penultimate residues of said 3′ terminus of said first strand are deoxyribonucleotides and the ultimate and penultimate residues of said 5′ terminus of said second strand are ribonucleotides.
190 . The isolated dsNA of claim 157 , wherein a nucleotide of said second or first oligonucleotide strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage.
191 . The isolated dsNA of claim 157 , wherein starting from the first nucleotide (position 1*) at the 3′ terminus of said second strand, positions 1*, 2*, and 3* from the 3′ terminus of said second strand are modified nucleotides.
192 . The isolated dsNA of claim 157 , wherein the first strand has a nucleotide sequence that is at least 80%, 90%, 95% or 100% complementary to the second strand nucleotide sequence.
193 . The isolated dsNA of claim 157 , wherein the 3′ terminus of said first strand and the 5′ terminus of said second strand are joined by a chemical linker.
194 . The isolated dsNA of claim 157 , wherein the dsNA is a Dicer substrate.
195 . The isolated dsNA of claim 157 , wherein the dsNA is a Dicer substrate that, upon endogenous Dicer processing, yields double-stranded nucleic acids of 19-23 nucleotides in length capable of reducing target gene expression in a mammalian cell.
196 . The isolated dsNA of claim 157 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.
197 . The isolated dsNA of claim 157 , wherein the dsNA reduces target gene expression in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.
198 . The isolated dsNA of claim 157 , wherein the dsNA, when introduced into a mammalian cell, reduces target gene expression in comparison to a reference dsRNA that does not possess a deoxyribonucleotide-deoxyribonucleotide base pair.
199 . The isolated dsNA of claim 157 , wherein the dsNA, when introduced into a mammalian cell, reduces target gene expression by at least 70% when transfected into said cell at a concentration selected from the group consisting of 1 nM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less and 10 pM or less.
200 . The isolated dsNA of claim 157 , wherein at least 50% of the ribonucleotide residues of said dsNA are unmodified ribonucleotides.
201 . The isolated dsNA of claim 157 , wherein at least 50% of the ribonucleotide residues of said second strand are unmodified ribonucleotides.
202 . The isolated dsNA of claim 157 , wherein the at least one of positions 24 to the 3′ terminal nucleotide residue of said first strand that is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand is an unmodified deoxyribonucleotide.
203 . The isolated dsNA of claim 202 , wherein both said at least one of positions 24 to the 3′ terminal nucleotide residue of said first strand that is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of said second strand and said deoxyribonucleotide of said second strand are unmodified deoxyribonucleotides.
204 . The isolated dsNA of claim 157 , wherein at least 50% of all deoxyribonucleotides of said dsNA are unmodified deoxyribonucleotides.
205 . The isolated dsNA of claim 157 , wherein the second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5′ terminal nucleotide residue of said first oligonucleotide strand and toward the 5′ end of said second strand, comprises alternating modified and unmodified nucleotide residues.
206 . The isolated dsNA of claim 157 , wherein at least one of positions 24 to the 3′ terminal nucleotide residue of said first strand is a phosphorothioate-modified nucleotide (PS-NA) That base pairs with a deoxyribonucleotide of said second strand
207 . The isolated dsNA of claim 157 , wherein the target RNA is KRAS.
208 . A method for reducing expression of a target gene in a cell, comprising: contacting a cell with an isolated double stranded NA (dsNA) as claimed in claim 157 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA.
209 . A method for reducing expression of a target gene in an animal, comprising: treating an animal with an isolated double stranded NA (dsNA) as claimed in claim 157 in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRNA.
210 . The method of claim 209 , wherein the dsNA possesses enhanced pharmacokinetics when compared to an appropriate control DsiRNA.
211 . The method of claim 209 , wherein the dsNA possesses enhanced pharmacodynamics when compared to an appropriate control DsiRNA.
212 . The method of claim 209 , wherein the dsNA possesses reduced toxicity when compared to an appropriate control DsiRNA.
213 . The method of claim 209 , wherein the dsNA possesses enhanced intracellular uptake when compared to an appropriate control DsiRNA.
214 . A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising the isolated double stranded NA (dsNA) of claim 157 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.
215 . A method of synthesizing the double stranded NA (dsNA) of claim 157 , comprising chemically or enzymatically synthesizing said dsNA.
216 . A kit comprising the dsNA of claim 157 and instructions for its use.