RNAi modulation of ApoB and uses thereof
View Patent ↗The invention relates to compositions and methods for modulating the expression of apolipoprotein B, and more particularly to the downregulation of apolipoprotein B by chemically modified oligonucleotides.
1. An iRNA agent comprising a sense strand having at least 17 contiguous nucleotides of sense strand sequence of iRNA agent number 35 (SEQ ID NO:5), and an antisense strand having at least 15 contiguous nucleotides of antisense sequence of iRNA agent number 35 (SEQ ID NO:6).
2. An iRNA agent comprising a sense strand and antisense strand each comprising a sequence of at least 19 nucleotides which is essentially identical to the sense or antisense strand sequence of iRNA agent number 35 (SEQ ID NOs: 5or 6), except that not more than 1 or 2 nucleotides per strand, respectively have been substituted by other nucleotides.
3. An iRNA agent selected from the group of iRNA agent number 66 (SEQ ID NOs:287, 288) and 67 (SEQ ID NOs:289, 290).
4. The iRNA agent of claim 1 , wherein the antisense RNA strand is 30 or fewer nucleotides in length, and duplex region of the iRNA agent is 17 to 30 nucleotide pairs in length.
5. The iRNA agent of claim 1 , wherein said iRNA agent reduces the amount of ApoB mRNA present in cultured mouse cells of hepatic origin.
6. The iRNA agent of claim 1 , comprising a modification that causes the iRNA agent to have increased stability in a biological sample.
7. The iRNA agent of claim 1 , comprising a phosphorothioate or a 2′-modified nucleotide.
8. The IRNA agent of claim 7 , comprising at least one 5′-uridine-adenine-3′ (5′-US-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′(5′-UG-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; at least one 5′-cytidine-adenin-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; or at least one 5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide.
9. The iRNA agent of claim 8 , wherein the 5′-most pyrimidines in all occurrences of sequence motif 5′-UA-3′, 5′-CA-3′, 5′-UU-3′, and 5′-UG-3′are 2′-modified nucleotides, or wherein all pyrimidines in the sense strand are 2′-modified nucleotides, and the 5′-most pyrimidines in all occurrences of the sequence motifs 5′-UA-3′and 5′-CA-3′, or wherein all pyrimidines in the sense strand are 2′-modified nucleotides, and the 5 ′-most pyrimidines in all occurrences of the sequence motifs 5′-UA-3′, 5′-CA -3′, 5′-UU-3′, and 5′- UG-3′ are 2′-modified nucleotides in the antisense strand.
10. The iRNA agent of claim 7 , wherein the 2′-modificaiton is chosen from the group of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-0-aminopropyl (2′-O-AP), 2′-0-dimethylaminoethyl (2′-O-DMAOE), 2′- dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O-N-methylacetamido (2′-O-NMA).
11. The iRNA agent of claim 1 , comprising a nucleotide overhang having 1 to 4 nucleotides.
12. The iRNA agent of claim 11 , wherein the nucleotide overhang has 2 or 3 unpaired nucleotides.
13. The iRNA agent of claim 11 , wherein the nucleotide overhang is at the 3′-end of the antisense strand of the iRNA agent.
14. The iRNA agent of claim 1 , comprising a cholesterol moiety.
15. The IRNA agent of claim 14 , wherein the cholesterol moiety is conjugated to the 3′-end of the sense strand of the iRNA agent.
16. The iRNA agent of claim 1 , where the iRNA agent is targeted for uptake by cells of the liver.
17. The iRNA agent of claim 1 , where the iRNA agent is targeted for uptake by cells of the gut.
18. A pharmaceutical composition, comprising:
a. an iRNA agent of claim 1 , and
b. a pharmaceutically acceptable carrier.
19. The iRNA agent of claim 1 , wherein the agent reduces the amount of ApoB mRNA in cultured human HepG2 cells after incubation with the agent by more than 60% compared to cells that have not been incubated with the agent, or reduces the amount of ApoB protein secreted into cell culture supernatant by more than 60%.
20. The iRNA agent of claim 1 , wherein the agent reduces the amount of ApoB mRNA in cultured human HepG2 cells after incubation with the agent by more than 70% compared to cells that have not been incubated with the agent, or reduces the amount of ApoB protein secreted into cell culture supernatant by more than 70%.
21. An iRNA agent comprising a sense strand and an antisense strand, each comprising a sequence of at least 19 nucleotides that are essentially identical to the sense strand or antisense strand sequence of iRNA agent number 35 (SEQ ID NOs: 5and 6), except that not more than 1 or 2 nucleotides per strand, respectively, have been substituted by other nucleotides, and wherein the agent retains the ability to inhibit ApoB expression in cultured human HepG2 cells, or the ability to reduce the amount of ApoB mRNA in murine liver cells of a C57B1/6 mouse by at least 20% in vivo after administration of 50 mg/kg body weight to 100 mg/kg body weight of the agent to the mouse.
22. The iRNA agent of claim 1 , wherein the antisense strand has at least 17 contiguous nucleotides of antisense sequence of iRNA agent number 35 (SEQ ID NO:6).