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 method for treating a platelet-mediated disorder comprising, administering to a host in need thereof a therapeutically effective amount of a GPVI ligand, wherein the GPVI ligand is a nucleic acid comprising a ligand nucleic acid sequence wherein the ligand nucleic acid sequence is characterized by a secondary structure that comprises 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 the fourth loop comprises a consensus sequence comprising UAA.
2. The method of claim 1 , wherein platelet-mediated disorder is selected from the group consisting of a vascular disorder, a cerebrovascular disorder, a platelet-mediated inflammatory disorder, a diabetes-related disorder, or a cancer.
3. The method of claim 2 , wherein the vascular disorder is selected from the group consisting of acute coronary syndromes, thrombosis, thromboembolism, peripheral vascular disease, and transient ischemic attack.
4. The method of claim 2 , wherein the cerebrovascular disorder is selected from the group consisting of transient ischemic attack, ischemic stroke, and embolism.
5. The method of claim 2 , wherein the platelet-mediated inflammatory disorder selected from the group consisting of arthritis, rheumatoid arthritis, psoriatic arthritis, reactive arthritis, inflammatory bowed disease, ankylosing spondylitis, and scleroderma.
6. The method of claim 2 , wherein the diabetes-related disorder is selected from the group consisting of diabetic retinopathy, diabetic vasculopathy, atherosclerosis, ischemic stroke, peripheral vascular disease, acute renal injury and chronic renal failure.
7. The method of claim 2 , wherein the cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, pancreatic cancer, brain cancer, bone cancer and liver cancer.
8. The method of claim 1 , further comprising administering to the host a nucleic acid modulator that specifically binds the GPVI ligand.
9. A method for modulating platelet function in a host in need thereof comprising administering to the host an effective amount of a GPVI ligand comprising
a ligand nucleic acid sequence wherein the ligand nucleic acid sequence is characterized by a secondary structure that comprises 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 the fourth loop comprises a consensus sequence comprising UAA.
10. The method of claim 9 , wherein the host is undergoing a cardiac intervention.
11. The method of claim 9 , further comprising administering to the host a nucleic acid modulator that specifically binds the GPVI ligand.
12. The method of claim 11 , wherein the modulator comprises a modulator nucleic acid sequence that is complementary to a portion of the GPVI ligand.
13. The method of claim 12 , wherein the modulator is selected from the nucleic acid sequences listed in Table 7.
14. The method of claim 12 , wherein the modulator comprises a modulator nucleic acid sequence that comprises 3′-AUU-5′, and which is capable of binding specifically to the fourth loop of the GPVI nucleic acid ligand.
15. The method of claim 14 , wherein the modulator comprises SEQ ID NO:84.
16. The method of claim 11 , wherein the ligand or modulator nucleic acid sequence comprises at least one modified nucleotide.
17. The method of claim 16 , wherein the at least one modified nucleotide is selected from 2′-O-methyl and 2′-fluoro.
18. The method of claim 11 , wherein the ligand or modulator is conjugated to a carrier.
19. The method of claim 18 , wherein the carrier is a hydrophilic moiety.
20. The method of claim 19 , wherein the hydrophilic moiety is polyethylene glycol (PEG) molecule.
21. The method of claim 11 , wherein the binding of the modulator to the GPVI ligand exposes a suicide position within the GPVI ligand, thereby disrupting the secondary structure of the GPVI ligand and leading to enhanced destruction of the nucleic acid GPVI ligand by nucleases.
22. The method of claim 9 , wherein the GPVI ligand comprises RB569, RB570 and RB571 from Table 6.
23. The method of claim 9 , wherein the effective amount reduces or inhibits platelet adhesion and/or aggregation.