Application of Nucleic Acid Aptamers and Scavengers for Thrombosis and Cancer

Sullenger, Bruce A

Gunaratne, Ruwan

2018-05-31T21:11:50Z

2020-04-26T08:17:15Z

2017

Pharmacology

Cardiovascular disease and cancer are the two leading causes of death in the U.S. Open heart surgery involving cardiopulmonary bypass (CPB) is performed in over a million patients worldwide in order to treat patients with severe ischemic cardiovascular disease, among other cardiac pathologies. Due to fulminant activation of the hemostatic system, CPB surgery requires administration of highly potent anticoagulation to prevent thrombosis during the procedure, followed by rapid neutralization to minimize the risk of post-operative bleeding. Since the 1950s, unfractionated heparin (UFH) has remained the standard anticoagulant for CPB surgery because of both its potency and reversibility with protamine. Unfortunately, UFH has several limitations that contribute to patient morbidity associated with CPB, creating an unmet clinical need for anticoagulant alternatives to UFH for CPB that can overcome its drawbacks while still maintaining robust potency and antidote-control. A similar clinical need exists for improved therapies that combat metastatic disease, which is ultimately responsible for the vast majority of cancer deaths, a fact that is particularly true for highly aggressive solid tumors such as pancreatic cancer.

Nucleic acid aptamers are an emerging class of therapeutics that are especially attractive as anticoagulants because their activity can be readily reversed by administration of either sequence specific antidotes comprised of complementary oligonucleotides or by “universal” antidotes that include certain cationic nucleic acid binding polymers (NABPs). Our lab has generated several antidote-controllable RNA aptamers that specifically inhibit individual coagulation factors, which have shown promising efficacy as anti-thrombotic agents in pre-clinical and clinical studies for indications other than CPB. Nevertheless, none of these aptamers when used alone can sufficiently match the anticoagulant intensity of UFH needed for CPB. 11F7t is one such aptamer which binds to exosites on both Factor (F)X and FXa and inhibits several procoagulant steps important for clot formation, but does not occlude the protease’s active site. As such, 11F7t’s mechanism of action is distinct from small molecule catalytic site inhibitors of FXa which are used clinically for oral thromboprophylaxis but are potent enough to facilitate anticoagulation during invasion procedures like CPB. In the first part of this work, we demonstrate that 11F7t and catalytic site inhibitors of FXa reciprocally and potently enhance anticoagulation in purified reaction mixtures and in plasma. Furthermore, such combinations prevent clot formation as effectively as UFH in human blood circulated within an extracorporeal oxygenator circuit that mimics CPB, while limiting thrombin generation and immunogenic platelet activation, which contribute to complications associated with UFH-facilitated CPB. Finally, we show that addition of GD-FXaS195A, a Gla-domainless FXa variant with an alanine substitution at the catalytic serine, can promptly neutralize the anticoagulant effects of both FXa inhibitors. GD-FXaS195A closely mimics a therapeutic (Andexanet Alfa) currently in late-stage clinical trials as a universal antidote specific for FXa inhibitors. Thus, our findings suggest promising avenues for developing improved alternatives to UFH for potent, antidote-controllable CPB anticoagulation.

In the second part of this work, we identify a novel application of NABPs for therapeutic inhibition of pancreatic cancer metastasis. Beyond their utility as universal antidotes for aptamers, our lab previously discovered that a subset of NABPs can also serve as anti-inflammatory agents by capturing extracellular nucleic acids and associated protein complexes that promote pathological activation of toll-like receptors (TLRs) in diseases such as systemic lupus erythematosus, sepsis, and influenza infection. Nucleic acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic acid-mediated activation of TLRs and invasion of PC tumor cells in vitro, but also can directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

https://hdl.handle.net/10161/16779

Pharmacology

Application of Nucleic Acid Aptamers and Scavengers for Thrombosis and Cancer

Dissertation

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