Over 80% of all strokes result from the presence of a thrombus occluding the cerebrovascular circulation. Definitive treatment of these strokes involves thrombolysis, that is, dissolution of the blood clot. Unfortunately, thrombolytic therapy is often incomplete and not uncommonly produces intracranial hemorrhage. Therefore, the broad, long-term objective of this proposal is to develop safer, more effective thrombolytic agents in order to reduce the number of deaths that result from stroke. As such, the overall goal of this research is to develop a thrombolytic agent that can have its activity reversed by a matched antidote. In this proposal, I will test the central hypothesis that reversible inhibition of the antifibrinolytic proteins plasminogen activator inhibitor-1 (PAI-1) and antiplasmin through aptamer-antidote pairs can produce safe and effective thrombolysis for treatment of stroke. To investigate this hypothesis, I will exploit the biochemical properties of RNA through the use of combinatorial chemistry to identify novel thrombolytic agents. In addition, I will utilize rational drug design to develop antidotes specific to these RNA molecules, resulting in a drug-antidote pair.
Specific aim 1 concerns isolation and characterization of nuclease-resistant RNA ligands called aptamers that bind to PAI-1 and antiplasmin. The SELEX technique will be used to isolate the aptamers, and nitrocellulose filter binding assays will be employed to determine the affinity of the aptamers for their protein targets.
In specific aim 2, those aptamers identified as having high affinity (Kd <10 nM) will undergo in vitro testing to determine their ability to functionally inhibit their protein targets.
Specific aim 3 involves optimization of the functional aptamers and design of their antidote oligonucleotides. Once developed, these aptamer-antidote pairs will also undergo functional testing to determine their ability to serve as reversible thrombolytic agents. Relevance to public health: Current treatment of stroke is often incomplete and not uncommonly results in bleeding into the brain. Therefore, this research aims at developing an effective drug to treat stroke and its matched reversal agent to improve its safety profile. The creation of this drug-antidote pair has the potential to reduce the amount of death and disability that results from stroke. ? ? ?
| Blake, Charlene M; Wang, Haichen; Laskowitz, Daniel T et al. (2011) A reversible aptamer improves outcome and safety in murine models of stroke and hemorrhage. Oligonucleotides 21:11-9 |
| Blake, Charlene M; Sullenger, Bruce A; Lawrence, Daniel A et al. (2009) Antimetastatic potential of PAI-1-specific RNA aptamers. Oligonucleotides 19:117-28 |
