5'-and 3'-capped aptamers and uses therefor
The invention provides compositions and methods for the treating disease using aptamers having 5′-5′ and 3′-3′ inverted nucleotide capped ends. In particular, the invention provides 5′-5′ and 3′-3′ capped anti-VEGF aptamers for the treatment of neovascularization-related diseases and disorders including age-related macular degeneration.
1 . An aptamer comprising 5′-5′ and 3′-3′ inverted nucleotide caps.
2 . The aptamer of claim 1 , wherein the aptamer is an RNA aptamer.
3 . The aptamer of claim 1 , wherein the aptamer is a DNA aptamer.
4 . An anti-VEGF aptamer comprising 5′-5′ and 3′-3′ inverted nucleotide caps.
5 . The anti-VEGF aptamer of claim 4 , wherein the aptamer is an RNA aptamer.
6 . The anti-VEGF aptamer of claim 4 , wherein the aptamer is a DNA aptamer.
7 . The anti-VEGF aptamer of claim 4 , comprising the sequence GAAGAAUUGG (SEQ ID NO 2), wherein each C, A, G and U is a naturally occurring or modified nucleoside.
8 . The anti-VEGF aptamer of claim 4 , comprising the sequence UUGGACGC (SEQ ID NO 3), wherein each C, A, G and U is a naturally occurring or modified nucleoside.
9 . The anti-VEGF aptamer of claim 4 , comprising the sequence GUGAAUGC (SEQ ID NO 4), wherein each C, A, G and U is a naturally occurring or modified nucleoside.
10 . The aptamer of claim 4 , further described by formula I:
X-5′-5′-CGGAAUCAGUGAAUGCUUAUACAUCCG-
(SEQ ID NO:1)
3′-3′-X
I
wherein C, G, A, and U represent their respective cytidylic, guanylic, adenylic, and uridylic acid nucleotides, X-5′-5′ is an inverted nucleotide capping the 5′ terminus of the aptamer and 3′-3′-X is an inverted nucleotide capping the 3′ terminus of the aptamer, and the remaining nucleotides are sequentially linked via 5′-3′ phosphodiester linkages.
11 . The aptamer of claim 10 , wherein each of said nucleotides, individually, comprise a 2′ ribosyl substituent selected from the group consisting of OH, H, O(C 1-10 alkyl), O(C 1-10 alkenyl), F, N 3 , and NH 2 .
12 . The aptamer of claim 11 , further described by formula II:
T d -5′-5′-C f G m G m A r A r U f C f A m G m U f G m A m A m U f
(SEQ ID NO 1)
G m C f U f U f A m U f A m C f A m U f C f C f G m 3′-3′-T d
II
wherein G m represents 2′-methoxyguanylic acid, A m represents 2′-methoxyadenylic acid, C f represents 2′-fluorocytidylic acid, U f represents 2′-fluorouridylic acid, A r represents riboadenylic acid, and T d represents deoxyribothymidylic acid.
13 . A pharmaceutical composition comprising an effective amount of an aptamer comprising 5′-5′ and 3′-3′ inverted nucleotide caps, together with a pharmaceutically acceptable carrier or diluent.
14 . The pharmaceutical composition of claim 13 , wherein the aptamer is an RNA aptamer.
15 . The pharmaceutical composition of claim 13 , wherein the aptamer is a DNA aptamer.
16 . A pharmaceutical composition comprising an effective amount of an anti-VEFG aptamer having 5′-5′ and 3′-3′ inverted nucleotide caps, together with a pharmaceutically acceptable carrier or diluent.
17 . The pharmaceutical composition of claim 16 , wherein the anti-VEGF aptamer is an RNA aptamer.
18 . The pharmaceutical composition of claim 16 , wherein the anti-VEGF aptamer is a DNA aptamer.
19 . The pharmaceutical composition of claim 16 , wherein the anti-VEGF aptamer comprises the sequence GAAGAAUUGG (SEQ ID NO 2), and each C, A, G and U is a naturally occurring or modified nucleoside.
20 . The pharmaceutical composition of claim 16 , wherein the anti-VEGF aptamer comprises the sequence UUGGACGC (SEQ ID NO 3), and each C, A, G and U is a naturally occurring or modified nucleoside.
21 . The pharmaceutical composition of claim 16 , wherein the anti-VEGF aptamer comprises the sequence GUGAAUGC (SEQ ID NO 4), and each C, A, G and U is a naturally occurring or modified nucleoside.
22 . The pharmaceutical composition of claim 16 , wherein the anti-VEGF aptamer is further described by formula I:
X-5′-5′-CGGAAUCAGUGAAUGCUUAUACAUCCG-
(SEQ ID NO:1)
3′-3′-X
I
wherein C, G, A, and U represent their respective cytidylic, guanylic, adenylic, and uridylic acid nucleotides, X-5′-5′ is an inverted nucleotide capping the 5′ terminus of the aptamer and 3′-3′-X is an inverted nucleotide capping the 3′ terminus of the aptamer, and the remaining nucleotides are sequentially linked via 5′-3′ phosphodiester linkages.
23 . The pharmaceutical composition of claim 22 , wherein each of said nucleotides, individually, comprise a 2′ ribosyl substituent selected from the group consisting of OH, H, O(C 1-10 alkyl), O(C 1-10 alkenyl), F, N 3 , and NH 2 .
24 . The pharmaceutical composition of claim 23 , wherein the anti-VEGF aptamer is further described by formula II:
T d -5′-5′-C f G m G m A r A r U f C f A m G m U f G m A m A m U f
(SEQ ID NO:1)
G m C f U f U f A m U f A m C f A m U f C f C f G m 3′-3′-T d
II
wherein G m represents 2′-methoxyguanylic acid, A m represents 2′-methoxyadenylic acid, C f represents 2′-fluorocytidylic acid, U f represents 2′-fluorouridylic acid, A r represents riboadenylic acid, and T d represents deoxyribothymidylic acid.
25 . A composition for the sustained release of an aptamer comprising an effective amount of an aptamer having 5′-5′ and 3′-3′ inverted nucleotide caps and a biocompatible polymer which allows for the release of the aptamer.
26 . The composition of claim 25 , wherein the aptamer is an RNA aptamer.
27 . The composition of claim 25 , wherein the aptamer is a DNA aptamer.
28 . A composition for the sustained release of an aptamer comprising an effective amount of an anti-VEGF aptamer having 5′-5′ and 3′-3′ inverted nucleotide caps and a biocompatible polymer which allows for the release of the aptamer.
29 . The composition of claim 28 , wherein the anti-VEGF aptamer is an RNA aptamer.
30 . The composition of claim 28 , wherein the anti-VEGF aptamer is a DNA aptamer.
31 . The composition of claim 28 , wherein the anti-VEGF aptamer comprises the sequence GAAGAAUUGG (SEQ ID NO 2), and each C, A, G and U is a naturally occurring or modified nucleoside.
32 . The composition of claim 28 , wherein the anti-VEGF aptamer comprises the sequence UUGGACGC (SEQ ID NO 3), and each C, A, G and U is a naturally occurring or modified nucleoside.
33 . The composition of claim 28 , wherein the anti-VEGF aptamer comprises the sequence GUGAAUGC (SEQ ID NO 4), and each C, A, G and U is a naturally occurring or modified nucleoside.
34 . The composition of claim 28 , wherein the anti-VEGF aptamer is further described by formula I:
X-5′-5′-CGGAAUCAGUGAAUGCUUAUACAUCCG-
(SEQ ID NO:1)
3′-3′-X
I
wherein C, G, A, and U represent their respective cytidylic, guanylic, adenylic, and uridylic acid nucleotides, X-5′-5′ is an inverted nucleotide capping the 5′ terminus of the aptamer and 3′-3′-X is an inverted nucleotide capping the 3′ terminus of the aptamer, and the remaining nucleotides are sequentially linked via 5′-3′ phosphodiester linkages.
35 . The composition of claim 34 , wherein each of said nucleotides, individually, comprise a 2′ ribosyl substituent selected from the group consisting of OH, H, O(C 1-10 alkyl), O(C 1-10 alkenyl), F, N 3 , and NH 2 .
36 . The composition of claim 35 , wherein the anti-VEGF aptamer is further described by formula II:
T d -5′-5′-C f G m G m A r A r U f C f A m G m U f G m A m A m U f
(SEQ ID NO:1)
G m C f U f U f A m U f A m C f A m U f C f C f G m 3′-3′-T d
II
wherein G m represents 2′-methoxyguanylic acid, A m represents 2′-methoxyadenylic acid, C f represents 2′-fluorocytidylic acid, U f represents 2′-fluorouridylic acid, A r represents riboadenylic acid, and T d represents deoxyribothymidylic acid.
37 . The composition of claim 28 , wherein said aptamer comprises from 0.1% (w/w) to 30% (w/w) of said composition.
38 . The composition of claim 37 , wherein said aptamer comprises from 0.1% (w/w) to 10% (w/w) of said composition.
39 . The composition of claim 38 , wherein said aptamer comprises from 0.5% (w/w) to 5% (w/w) of said composition.
40 . The composition of claim 28 , further comprising a stabilizing agent selected from the group consisting of saccharides, poly alcohols, proteins and hydrophilic polymers.
41 . The composition of claim 28 , wherein said biocompatible polymer is degradable under physiological conditions.
42 . The composition of claim 41 , wherein said degradable polymer is selected from the group consisting of polycarbonates, polyanhydrides, polyamides, polyesters, polyorthoesters, bioerodable hydrogels, and copolymers and mixtures thereof.
43 . The composition of claim 42 , wherein said polyester is selected from the group consisting of poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), polycaprolactone, blends thereof, and copolymers thereof.
44 . The composition of claim 42 , wherein said polymer comprises poly(lactic acid-co-glycolic acid).
45 . The composition of claim 28 , wherein said biocompatible polymer is a non-degradable polymer.
46 . The composition of claim 45 , wherein the non-degradable polymer is a silicone derivative.
47 . The composition of claim 45 , wherein the non-degradable polymer is selected from the group consisting of polysaccharides, polyether, vinyl polymer, polyurethane, cellulose-based polymer, and polysiloxane.
48 . The composition of claim 47 , wherein the polyether is selected from the group consisting of poly (ethylene oxide), poly (ethylene glycol), and poly (tetramethylene oxide).
49 . The composition of claim 47 , wherein the vinyl polymer is selected from the group consisting of polyacrylates, acrylic acids, poly (vinyl alcohol), poly (vinyl pyrolidone), and poly (vinyl acetate).
50 . The composition of claim 47 , wherein the cellulose-based polymer is selected from the group consisting of cellulose, alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, cellulose esters, nitrocellulose, and cellulose acetates.
51 . The composition of claim 28 , wherein said composition is a solid particulate having an average diameter of less than 400 μm.
52 . The composition of claim 51 , wherein said composition is a solid particulate having an average diameter of less than 200 μm.
53 . The composition of claim 52 , wherein said composition is a solid particulate having an average diameter of less than 100 μm.
54 . A composition for the sustained release of an aptamer comprising an effective amount of an anti-VEGF aptamer having 5′-5′ and 3′-3′ inverted caps and a biocompatible polymer which is degradable under physiological conditions.
55 . The composition of claim 54 , wherein the half-life for the release of said aptamer on the sclera of an eye is greater than 1 month.
56 . The composition of claim 55 , wherein the half-life for the release of said aptamer on the sclera of an eye is greater than 2 months.
57 . The composition of claim 56 , wherein the half-life for the release of said aptamer on the sclera of an eye is greater than 4 months.