Modified oligonucleotides for telomerase inhibition
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IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12N-015/113
C07C-233/18
A61K-047/48
C07H-021/02
C07H-021/04
출원번호
US-0714732
(2015-05-18)
등록번호
US-9657296
(2017-05-23)
발명자
/ 주소
Gryaznov, Sergei
Pongracz, Krisztina
출원인 / 주소
Geron Corporation
대리인 / 주소
Foulds, Glenn J.
인용정보
피인용 횟수 :
1인용 특허 :
49
초록▼
Compounds comprising an oligonucleotide moiety covalently linked to a lipid moiety are disclosed. The oligonucleotide moiety comprises a sequence that is complementary to the RNA component of human telomerase. The compounds inhibit telomerase activity in cells with a high potency and have superior c
Compounds comprising an oligonucleotide moiety covalently linked to a lipid moiety are disclosed. The oligonucleotide moiety comprises a sequence that is complementary to the RNA component of human telomerase. The compounds inhibit telomerase activity in cells with a high potency and have superior cellular uptake characteristics.
대표청구항▼
1. A method of synthesizing a lipid conjugated oligonucleotide comprising at least 5 contiguous nucleoside subunits complementary to the RNA component of human telomerase each subunit joined by a N3′→P5′ thiophosphoramidate linkage, the method comprising: (a) deprotecting and reacting a lipid contai
1. A method of synthesizing a lipid conjugated oligonucleotide comprising at least 5 contiguous nucleoside subunits complementary to the RNA component of human telomerase each subunit joined by a N3′→P5′ thiophosphoramidate linkage, the method comprising: (a) deprotecting and reacting a lipid containing solid support with a first 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to produce a first nucleoside attached to the lipid containing solid support through a phosphite linkage, wherein after the deprotecting the lipid containing solid support comprises the structure wherein Suc is a succinyl group that is attached to the solid support and R″ is the carbon chain of a fatty acid lipid or a sterol; (b) sulfurizing the phosphite linkage to form a thiophosphate linkage;(c) deprotecting the protected 3′-amino group of the first aminonucleoside to form a free 3′-amino group;(d) reacting the free 3′-amino group with a second 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to form an internucleoside N3′→P5′ phosphoramidite linkage; and(e) sulfurizing the internucleoside N3′→P5′ phosphoramidite linkage to form a N3′→P5′ thiophosphoramidate linkage; and(f) repeating steps (c) through (e) to produce the lipid conjugated oligonucleotide. 2. The method of claim 1, further comprising releasing the lipid conjugated oligonucleotide bound to the solid support to produce a free lipid conjugated oligonucleotide, or a salt thereof. 3. The method of claim 1, wherein the lipid containing solid support comprises the structure: where R is a solid support, DMT is dimethoxytrityl and n is 6-20 such that the carbon chain —C(O)(CH2)nCH3 comprises from C8-C22 carbons. 4. The method of claim 2, wherein the free lipid containing oligonucleotide comprises the structure O-(x-L), wherein L comprises a lipid moiety and x comprises an amino glycerol linker, and a linked phosphate group, wherein the amino glycerol linker comprises the structure 5. The method of claim 4, wherein the lipid moiety is selected from the group consisting of substituted fatty acids, and unsubstituted fatty acids. 6. The method of claim 5, wherein the lipid moiety is a fatty acid substituted with fluorine. 7. The method of claim 4, wherein the oligonucleotide comprises an oligonucleotide sequence selected from the group consisting of: TAGGGTTAGACAA;GTTAGGGTTAG;GTTAGGGTTAGAC;GTTAGGGTTAGACAA;GGGTTAGAC;CAGTTAGGG; orCAGTTAGGGTTAGwherein the internucleoside linkages of the oligonucleotide sequence are N3′→P5′ thiophosphoramidate linkages. 8. The method of claim 4, wherein (x-L) is selected from the group consisting of 5′-cholesterylamido-aminoglycerol-thiophosphate, 5′-palmitoylamido-aminoglycerol-thiosphosphate 5′-C11-teflon-aminoglycerol-thiophosphate and 5′-C13-teflon-aminoglycerol-thiophosphate. 9. The method of claim 8, wherein the oligonucleotide comprises TAGGGTTAGACAA and wherein the (x-L) is 5′palmitoylamido-aminoglycerol-thiosphosphate. 10. The method of claim 1, further comprising conjugating the 3′ terminus of the oligonucleotide with a second lipid moiety. 11. A method of synthesizing a lipid conjugated oligonucleotide comprising at least 5 contiguous nucleoside subunits complementary to the RNA component of human telomerase each subunit joined by a N3′→P5′ thiophosphoramidate linkage, the method comprising: (a) deprotecting a protected 3′-amino group of a first nucleoside attached to a lipid containing solid phase support, the first nucleoside having a protected 3′ amino group and a 5′ phosphate conjugated to the lipid containing solid phase support, wherein said deprotecting forms a free 3′-amino group of a first nucleoside attached to a lipid containing solid phase support comprising the structure: wherein:Suc is a succinyl group that is attached to the solid phase support (R);R″ is the carbon chain of a fatty acid lipid or a sterolX is O or S;Y is H; andB is the nucleobase of the first nucleoside;(b) reacting the free 3′-amino group with a second 3′-protected aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer to form an internucleoside N3′→P5′ phosphoramidite linkage; and(c) sulfurizing the internucleoside N3′→P5′ phosphoramidite linkage to form a N3′→P5′ thiophosphoramidate linkage; and(d) repeating steps (a) through (c) to produce the lipid conjugated oligonucleotide. 12. A method of synthesizing a lipid conjugated oligonucleotide, the method comprising conjugating a terminus of an oligonucleotide comprising the sequence TAGGGTTAGACAA with a lipid conjugate comprising the structure (x-L) wherein (x-L) is 5′palmitoylamido-aminoglycerol-thiosphosphate, wherein the internucleoside linkages of the oligonucleotide are N3′→P5′ thiophosphoramidate linkages. 13. The method of claim 11, further comprising conjugating the 3′ terminus of the oligonucleotide with a second lipid moiety. 14. The method of claim 11, wherein the lipid moiety is selected from the group consisting of substituted and unsubstituted fatty acids. 15. The method of claim 14, wherein the lipid moiety is a fatty acid substituted with fluorine. 16. The method of claim 11, wherein the oligonucleotide comprises an oligonucleotide sequence selected from the group consisting of: TAGGGTTAGACAA;GTTAGGGTTAG;GTTAGGGTTAGAC;GTTAGGGTTAGACAA;GGGTTAGAC;CAGTTAGGG;orCAGTTAGGGTTAG. 17. The method of claim 11, wherein the lipid conjugated oligonucleotide comprises the structure (x-L) that is selected from the group consisting of 5′-cholesterylamido-aminoglycerol-thiophosphate, 5′-palmitoylamido-aminoglycerol-thiosphosphate, 5′-C11-teflon-aminoglycerol-thiophosphate, 5′-C13-teflon-aminoglycerol-thiophosphate, and 5′-stearoylamido-aminoglycerol-thiophosphate. 18. The method of claim 17, wherein the oligonucleotide comprises TAGGGTTAGACAA and wherein (x-L) is 5′palmitoylamido-aminoglycerol-thiosphosphate. 19. A lipid containing solid support comprising the structure: wherein R is a solid support, DMT is dimethoxytrityl and n is 6-20 such that the carbon chain —C(O)(CH2)nCH3 comprises from C8-C22 carbons. 20. A method of preparing a modified lipid containing solid support, the method comprising: reacting a 3-amino-1,2-propanediol that comprises a 3-amino group, a 1-hydroxy group and a 2-hydroxy group with a fatty acid; dimethoxytritylating the 1-hydroxy group; andsuccinylating the 2-hydroxy group; to produce a 3-fatty acid amido-1-dimethoxytrityl-2-succinyloxy-propane; andreacting the 3-fatty acid amido-1-dimethoxytrityl-2-succinyloxy-propane with a support to produce the modified lipid containing solid support. 21. The method of claim 20, wherein: the fatty acid is palmitic acid,the 3-fatty acid amido-1-dimethoxytrityl-2-succinyloxy-propane is 3-palmitoylamino-1-dimethoxytrityl-2-succinyloxy-propane, andthe modified lipid containing solid support comprises the structure: wherein R is a solid support, DMT is dimethoxytrityl and n is 14.
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이 특허에 인용된 특허 (49)
Fung Steven (Palo Alto CA) Woo Sam L. (Redwood City CA) Smith Lloyd M. (South Pasadena CA), Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof.
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Letsinger Robert L. (Wilmette IL) Gryaznov Sergei M. (San Mateo CA), Oligodeoxyribonucleotides including 3′-aminonucleoside-phosphoramidate linkages and terminal 3′-amino groups.
Gryaznov Sergei M. (San Mateo CA) Schultz Ronald G. (Fremont CA) Chen Jer-kang (Palo Alto CA), Oligonucleotide N3′-P5′phosphoramidates: hybridization and nuclease resistance properties.
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Gryaznov Sergei M. (San Mateo CA) Schultz Ronald G. (Fremont CA) Chen Jer-kang (Palo Alto CA), Oligonucleotide N3→P5′phosphoramidates: triplex DNA formation.
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