IPC분류정보
국가/구분 |
United States(US) Patent
등록
|
국제특허분류(IPC7판) |
|
출원번호 |
US-0917680
(2006-06-08)
|
등록번호 |
US-8252756
(2012-08-28)
|
국제출원번호 |
PCT/US2006/022325
(2006-06-08)
|
§371/§102 date |
20090403
(20090403)
|
국제공개번호 |
WO2006/138145
(2006-12-28)
|
발명자
/ 주소 |
- Mirkin, Chad A.
- Rosi, Nathaniel L.
- Thaxton, C. Shad
- Giljohann, David A.
|
출원인 / 주소 |
|
대리인 / 주소 |
Marshall, Gerstein & Borun LLP
|
인용정보 |
피인용 횟수 :
4 인용 특허 :
183 |
초록
Materials and methods for regulating gene expression using nanoparticles functionalized with antisense oligonucleotides are provided.
대표청구항
▼
1. A method of inhibiting expression of a gene product comprising the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said oligonucleotide being covalently bound to an inorganic nanoparticle,
1. A method of inhibiting expression of a gene product comprising the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said oligonucleotide being covalently bound to an inorganic nanoparticle, wherein said nanoparticle does not comprise a targeting peptide, wherein hybridizing between said polynucleotide and said oligonucleotide, in a buffer and in the absence of a transfection reagent, occurs over a length of said polynucleotide with a degree of complementarity sufficient to inhibit expression of said gene product, wherein the expression is inhibited by at least 5% compared to expression in the absence of the oligonucleotide, and wherein said nanoparticle is not a dendrimer. 2. The method of claim 1 wherein expression of said gene product is inhibited in vitro or in vivo. 3. The method of claim 1 wherein said nanoparticle is metallic or is a colloidal metal, or is selected from the group consisting of a gold nanoparticle, a silver nanoparticle, a platinum nanoparticle, an aluminum nanoparticle, a palladium nanoparticle, a copper nanoparticle, a cobalt nanoparticle, an indium nanoparticle, and a nickel particle. 4. The method of claim 1 wherein said oligonucleotide is bound to said nanoparticle through one or more sulfur linkages. 5. The method of claim 1 wherein said oligonucleotide is about 5 to about 100 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 80 nucleotides in length, about 5 to about 70 nucleotides in length, about 5 to about 60 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, or about 5 to about 10 nucleotides in length. 6. The method of claim 1 wherein said oligonucleotide is a DNA oligonucleotide or an RNA oligonucleotide. 7. The method of claim 1 wherein said oligonucleotide is 100% complementary to said polynucleotide, greater than 95% complementary to said polynucleotide, greater than 90% complementary to said polynucleotide, greater than 80% complementary to said polynucleotide, greater than 75% complementary to said polynucleotide, greater than 70% complementary to said polynucleotide, greater than 65% complementary to said polynucleotide, greater than 60% complementary to said polynucleotide, greater than 55% complementary to said polynucleotide, or greater than 50% complementary to said polynucleotide. 8. The method of claim 1 wherein said oligonucleotide includes at least one modified internucleotide linkage selected from the group consisting of a peptide nucleic acid linkage, a phosphorothioate linkage, a morpholino linkage, a methylphosphonate linkage, or a sulfonyl linkage. 9. The method of claim 1 wherein said oligonucleotide includes at least one modified nucleic acid sugar moiety or at least one modified nucleic acid. 10. The method of claim 1 wherein said oligonucleotide is bound to said nanoparticle through a 5′ linkage or a 3′ linkage. 11. The method of claim 1 wherein said oligonucleotide comprises a tandem repeat of identical nucleotide sequences, and wherein said tandem repeat comprises two identical nucleotide sequences, three identical nucleotide sequences, four identical nucleotide sequences, five identical nucleotide sequences, or five or more identical nucleotide sequences. 12. The method of claim 11 wherein said identical nucleotide sequences in said tandem repeat are separated by a nucleotide spacer between each identical sequence. 13. The method of claim l wherein said oligonucleotide is bound through a spacer to said nanoparticle. 14. The method of claim 13 wherein said spacer is an organic moiety. 15. The method of claim 14 wherein said organic moiety is a polymer. 16. The method of claim 15 wherein said polymer is a water-soluble polymer, a nucleic acid, a polypeptide, or an oligosaccharide. 17. The method of claim 1 wherein said nanoparticle is bound to at least two oligonucleotides having different sequences. 18. The method of claim 17 wherein said different sequences hybridize to different regions on the same polynucleotide or to different polynucleotides. 19. The method of claim 1 wherein said polynucleotide is a mRNA encoding said gene product and translation of said gene product is inhibited. 20. The method of claim 1 wherein said polynucleotide is DNA in a gene encoding said gene product and transcription of said gene product is inhibited. 21. The method of claim 20 wherein said DNA encodes said gene product or is complementary to a sequence that encodes said gene product. 22. The method of claim 1 wherein said polynucleotide is a bacterial polynucleotide. 23. The method of claim 22 wherein said bacterial polynucleotide is bacterial genomic DNA or is RNA transcribed from bacterial genomic DNA. 24. The method of claim 1 wherein said polynucleotide is a viral polynucleotide. 25. The method of claim 24 wherein said viral polynucleotide is viral genomic RNA, viral genomic DNA, or RNA transcribed from viral genomic DNA. 26. The method of claim 1 wherein said polynucleotide is a fungal polynucleotide. 27. The method of claim 26 wherein said fungal polynucleotide is fungal genomic DNA or is RNA transcribed from fungal genomic DNA. 28. The method of claim 1 wherein expression of said gene product is inhibited by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. 29. The method of claim 1 wherein said nanoparticle ranges from about 1 nm to about 250 nm in mean diameter, about 1 nm to about 240 nm in mean diameter, about 1 nm to about 230 nm in mean diameter, about 1 nm to about 220 nm in mean diameter, about 1 nm to about 210 nm in mean diameter, about 1 nm to about 200 nm in mean diameter, about 1 nm to about 190 nm in mean diameter, about 1 nm to about 180 nm in mean diameter, about 1 nm to about 170 nm in mean diameter, about 1 nm to about 160 nm in mean diameter, about 1 nm to about 150 nm in mean diameter, about 1 nm to about 140 nm in mean diameter, about 1 nm to about 130 nm in mean diameter, about 1 nm to about 120 nm in mean diameter, about 1 nm to about 110 nm in mean diameter, about 1 nm to about 100 nm in mean diameter, about 1 nm to about 90 nm in mean diameter, about 1 nm to about 80 nm in mean diameter, about 1 nm to about 70 nm in mean diameter, about 1 nm to about 60 nm in mean diameter, about 1 nm to about 50 nm in mean diameter, about 1 nm to about 40 nm in mean diameter, about 1 nm to about 30 nm in mean diameter, or about 1 nm to about 20 nm in mean diameter, or about 1 nm to about 10 nm in mean diameter. 30. The method of claim 1 wherein said oligonucleotide is bound to said nanoparticle at a surface density of at least 10 pmol/cm2, at least 15 pmol/cm2, at least 20 pmol/cm2, at least 10 pmol/cm2, at least 25 pmol/cm2, at least 30 pmol/cm2, at least 35 pmol/cm2, at least 40 pmol/cm2, at least 45 pmol/cm2, or at least 50 pmol/cm2. 31. The method of claim 1 wherein expression of said gene product is associated with a disease state. 32. The method of claim 1 wherein said nanoparticle is optionally labeled. 33. The method of claim 1 wherein said nanoparticle further comprises a targeting molecule. 34. The method of claim 1 wherein said polynucleotide is a mitochondrial polynucleotide. 35. The method of claim 1 wherein packing density of the oligonucleotides on the surface of the nanoparticle is sufficient to result in cooperative behavior between said nanoparticles. 36. The method of claim 35 wherein the packing density of the oligonucleotides on the surface of the nanoparticle is greater than about 7000 oligonucleotides per square micron of nanoparticle surface. 37. The method of claim 35 wherein cooperative behavior between the nanoparticles increases the strength of the binding between the oligonucleotide and the polynucleotide, increases resistance of the oligonucleotide to degradation, increases resistance of the oligonucleotide-polynucleotide complex to degradation, or increases resistance of the oligonucleotide to degradation by a nuclease. 38. The method of claim 1 wherein said polynucleotide is an inhibitory RNA (RNAi) that performs a regulatory function. 39. The method of claim 38 wherein the RNAi is selected from the group consisting of a small inhibitory RNA (siRNA), an RNA that forms a triplex with double stranded DNA, and a ribozyme. 40. The method of claim 1 wherein the oligonucleotide is complementary to a regulatory region of the polynucleotide. 41. The method of claim 1 wherein the oligonucleotide is released from the nanoparticle after the nanoparticle enters a cell. 42. A method of inhibiting expression of a gene product comprising the step of hybridizing a polynucleotide encoding said gene product with one or more oligonucleotides complementary to all or a portion of said polynucleotide, said oligonucleotide being covalently bound to a nanoparticle, wherein said nanoparticle does not comprise a targeting peptide, wherein hybridizing between said polynucleotide and said oligonucleotide, in a buffer and in the absence of a transfection reagent, occurs over a length of said polynucleotide with a degree of complementarity sufficient to inhibit expression of said gene product, wherein the expression is inhibited by at least 5% compared to expression in the absence of the oligonucleotide, and wherein said nanoparticle is not a dendrimer or polyethylene.
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