RNA interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional siNA)
View Patent ↗The present invention concerns methods and nucleic acid based reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, veterinary, agricultural, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to multifunctional short interfering nucleic acid (multifunctional siNA) molecules that modulate the expression of one or more genes in a biologic system, such as a cell, tissue, or organism via RNA interference (RNAi). The bifunctional short interfering nucleic acid (multifunctional siNA) molecules of the invention can target more than one regions of nucleic acid sequence in a single target nucleic acid molecule or can target regions of nucleic acid sequence in differing target nucleic acid molecules. The self multifunctional siNA molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.
1. A multifunctional siNA molecule of Formula I:
5′- p -XZX′-3′
3′-Y′ZY- p -5′
wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ independently comprise an oligonucleotide of length between 24 and 38 nucleotides, XZ comprises a nucleic acid sequence that is complementary to a first target nucleic acid sequence, YZ comprises an oligonucleotide comprising nucleic acid sequence that is complementary to a second target nucleic acid sequence, Z comprises nucleotide sequence of length 1 to 24 nucleotides that is complementary between regions XZ and YZ, X comprises nucleotide sequence of length 1 to 21 nucleotides that is complementary to nucleotide sequence present in region Y′, Y comprises nucleotide sequence of length 1 to 21 nucleotides that is complementary to nucleotide sequence present in region X′, p comprises a terminal phosphate group that can independently be present or absent, and wherein each said XZ and said YZ are independently of length sufficient to stably interact with said first and said second target nucleic acid sequence, respectively, or a portion thereof.
2. The siNA molecule of claim 1 , wherein said siNA comprises a 3′-terminal cap moiety.
3. The siNA molecule of claim 2 , wherein said terminal cap moiety is an inverted deoxyabasic moiety.
4. The siNA molecule of claim 2 , wherein said terminal cap moiety is an inverted deoxynucleotide moiety.
5. The siNA molecule of claim 2 , wherein said terminal cap moiety is a dinucleotide moiety.
6. The siNA molecule of claim 5 , wherein said dinucleotide is dithymidine (TT).
7. The siNA molecule of claim 1 , wherein said siNA molecule comprises no ribonucleotides.
8. The siNA molecule of claim 1 , wherein said siNA molecule comprises ribonucleotides.
9. The siNA molecule of claim 1 , wherein any purine nucleotide in said siNA is a 2′-0-methyl pyrimidine nucleotide.
10. The siNA molecule of claim 1 , wherein any purine nucleotide in said siNA is a 2′-deoxy purine nucleotide.
11. The siNA molecule of claim 1 , wherein any pyrimidine nucleotide in said siNA is a 2′-deoxy-2′-fluoro pyrimidine nucleotide.
12. The siNA molecule of claim 1 , wherein said siNA molecule comprises 3′-nucleotide overhangs.
13. The siNA molecule of claim 12 , wherein said 3′-overhangs comprise about 1 to about 4 nucleotides.
14. The siNA molecule of claim 13 , wherein said nucleotides comprise deoxynucleotides.
15. The siNA molecule of claim 14 , wherein said deoxynucleotides are thymidine nucleotides.
16. A composition comprising the siNA molecule of claim 1 in a pharmaceutically acceptable carrier or diluent.