Nucleic acid modulators of glycoprotein VI
The present invention relates, in general, to a pharmacologic system to modulate the biology of platelets based upon a nucleic acid ligand that can interact with and modulate the activity of platelet glycoprotein GPVI to regulate platelet function. These nucleic acid ligands are also actively reversible using a modulator that inhibits the activity of the nucleic acid ligand to neutralize this pharmacologic effect and thereby restore GPVI function, including collagen binding, platelet adhesion, collagen-induced platelet activation, and collagen-induced platelet aggregation. The invention further relates to compositions comprising the nucleic acid ligand, the ligand and a modulator, methods to generate the nucleic acid ligand and its modulator, as well as methods of using these agents and compositions in medical therapeutic and diagnostic procedures.
1. A GPVI ligand comprising a first isolated nucleic acid sequence, wherein said GPVI ligand binds glycoprotein VI (GPVI) and wherein said GPVI ligand comprises a secondary structure comprising in a 5′ to 3′ direction a first stem, a first loop, a second stem, a second loop, a third loop, a third stem and a fourth loop; and wherein said fourth loop comprises UAA.
2. A GPVI ligand comprising a first isolated nucleic acid sequence, wherein said GPVI ligand binds glycoprotein VI (GPVI) and wherein said GPVI ligand comprises a secondary structure comprising in a 5′ to 3′ direction a first stem, a first loop, a second stem, a spacer, a third loop, a third stem and a fourth loop; and wherein said fourth loop comprises UAA.
3. The GPVI ligand of claim 1 , wherein the first nucleic acid comprises at least one modified nucleotide.
4. The GPVI ligand of claim 1 , conjugated to a carrier.
5. The GPVI ligand of claim 4 , wherein the carrier is a hydrophilic moiety.
6. The GPVI ligand of claim 5 , wherein the hydrophilic moiety is a polyethylene glycol (PEG) molecule.
7. The GPVI ligand of claim 1 , comprising a modified phosphate backbone.
8. The GPVI ligand of claim 2 , comprising SEQ ID NO:69, wherein the spacer is incorporated between the 2′O-Methyl G at position 11 and the 2′O-Methyl C at position 12 of SEQ ID NO:69.
9. The GPVI ligand of claim 8 , comprising a modified phosphate backbone.
10. The GPVI ligand of claim 2 , wherein said GPVI ligand is selected from the group consisting of RB569, RB570, and RB571 as described in Table 6.
11. A pharmaceutical composition comprising the GPVI ligand of claim 1 .
12. The GPVI ligand of claim 2 , wherein the first nucleic acid comprises at least one modified nucleotide.
13. The GPVI ligand of claim 2 , conjugated to a carrier.
14. The GPVI ligand of claim 13 , wherein the carrier is a hydrophilic moiety.
15. The GPVI ligand of claim 14 , wherein the hydrophilic moiety is a polyethylene glycol (PEG) molecule.
16. The GPVI ligand of claim 2 , wherein the spacer is a glycol spacer.
17. The GPVI ligand of claim 2 , wherein said GPVI ligand comprises SEQ ID NO:89.
18. The GPVI ligand of claim 2 , wherein said GPVI ligand comprises SEQ ID NO:90.
19. The GPVI ligand of claim 2 , wherein said GPVI ligand comprises SEQ ID NO:91.
20. The GPVI ligand of claim 2 , wherein said GPVI ligand consists of SEQ ID NO:89.
21. The GPVI ligand of claim 2 , wherein said GPVI ligand consists of SEQ ID NO:90.
22. The GPVI ligand of claim 2 , wherein said GPVI ligand consists of SEQ ID NO:91.
23. A pharmaceutical composition comprising the GPVI ligand of claim 2 .