Browsing by Author "Sullenger, Bruce A"

Current standard cancer treatments such as chemotherapeutics, and radiation therapy are nearly as likely to kill the patient as cure the cancer. Therapies that have such a narrow window of efficacy are necessary for the treatment of aggressive diseases, but safer alternatives must be created. By discovering novel therapeutics that target specific disease processes within specific diseased cells, while leaving healthy cells unaltered, we can improve the lives of millions of cancer sufferers and their families. A therapeutic's window of efficacy can be measured by the therapeutic index. For many anti-cancer therapeutics, the therapeutic index is very small, the dose of treatment that kills cancer cells and shrinks tumors is nearly the dose that causes toxicity. In cancer patients, this toxicity causes many serious conditions such as gastrointestinal distress, organ damage, and death.

Recently, the model of cancer treatment has evolved from non-specific cytotoxic agents to more selective therapeutics that target cellular processes necessary for cancer cell survival. If a therapy can be targeted to selectively bind and internalize targeted cells, its toxicity would only impact the targeted cells and healthy cells in the immediate vicinity, which would greatly reduce the toxic effects on the rest of the body. Targeting cancer cells can be done through cancer biomarkers, which are cell surface proteins, expressed exclusively, or are much more abundant on the surface of cancer cells than on somatic cells.

Advances in antibody and aptamer technology have enabled researchers to design those molecules to bind specifically to cancer cells, and deliver drugs that alter specific cellular processes. An aptamer designed to bind PSMA, a prostate cancer biomarker, only bound to a specific subset of cancer cells, and delivered a therapeutic siRNA that prevented a specific survival process from occurring. While this technology is promising, it is currently limited to targeting small subsets of cancer types. To generate an aptamer therapeutic that would have greater utility and efficacy, I have examined the properties of a nucleolin aptamer-mediated delivery system that targets multiple types of cancer cells, and delivers various oligonucleotide therapeutics.

The nucleolin aptamer targeted cancer cells by binding to membrane–associated nucleolin. Nucleolin, a conserved protein found in all eukaryotes, shuttles from the nucleus, through the cytoplasm to the cell membrane. Cancer cells express a far greater amount of membrane–associated nucleolin than somatic cells, making nucleolin an ideal cancer biomarker. The shuttling, and oligonucleotide binding attributes of the protein enable it to deliver aptamer chimeras from the cell surface to the nucleus. Therefore the nucleolin aptamer has unique access to the nuclei of cancer cells, and can deliver therapeutic oligonucleotide cargoes through nucleolin binding.

The nucleolin aptamer delivered splice–switching oligonucleotides, a form of antisense technology, improving their efficacy, and potentially increasing their therapeutic viability. The ability to deliver antisense oligonucleotides to the nuclei of cancer cells has the potential for other therapeutic possibilities including the inhibition of transcription with antisense triplexes.

The nucleolin aptamer can also deliver therapeutic aptamers. The nucleolin aptamer–β–arrestin aptamer chimera prevented the stem cell renewal phenotype necessary for leukemia progression in human patient tissue samples. The ability to effectively deliver therapeutic aptamers may lead to clinical applications for many of the aptamers that have been selected against intracellular targets including transcriptional activators.

Oligonucleotide research continues to advance our understanding of potentially therapeutic oligonucleotides. Long non–coding RNAs for example, may impact epigenetics, and transcription. Additionally, locked nucleic acids have been developed to improve binding affinity, thus increasing the efficacy of antisense oligonucleotides. In order to bring these discoveries into the clinic, they must be safely and specifically delivered to their target cells.

This work demonstrated that the nucleolin aptamer could deliver oligonucleotide therapeutics to specific cancer cells. Nucleolin aptamer chimeras have the potential to develop into safe and effective cancer therapies, thus improving the treatment options for cancer sufferers.

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.

Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) are two essential tools for cell separation in research and medicine. Antibodies, the gold standard in both of these methods, are effective ligands for cell-surface biomarkers, but their irreversible binding precludes a wide variety of downstream medical and experimental applications. Aptamers – nucleic acid ligands with a defined three-dimensional structure that enables them to bind a molecular target with a high degree of specificity – offer a viable alternative for this particular obstacle because their RNA- or DNA-based chemistry enables their removal from cellular targets. In these studies, we present examples of successful sorting of cells and removal of the targeting aptamers with MACS and FACS using both the previously-published antisense-based method of post-sorting aptamer removal and a more general approach using nuclease-based digestion of targeting aptamers on the cell surface after cell isolation. We believe this work can be used in a number of potential post-sorting applications where targeting ligands or attached magnetic or fluorescent moieties could interfere with experimental or clinical results.

Coagulation and inflammation are intimately linked processes that protect the body from a wide range of insults. However, dysregulation of this system contributes to morbidity and mortality in infection, autoimmune disease, and countless other disease processes. To successfully intervene on this axis, we need to understand the molecular drivers of thromboinflammation and design biocompatible pharmaceuticals to combat them. Towards that effort, this dissertation first explores the clinical landscape of COVID-19, with specific focus on characterization of the molecular drivers of COVID-19-associated coagulopathy. Through analysis of coagulative profiles and clinical data, fibrinolytic suppression and endothelial injury are identified as primary drivers of thrombosis and respiratory distress in COVID-19. Next, this dissertation describes efforts to reduce the toxicity of the anti-inflammatory scavenging polymer polyamidoamine (PAMAM). We show that the density of cationic surface charges underpins the direct cellular and systemic toxicity of these polymers and present novel PAMAM variants with a mix of cationic and neutral surface groups that resolve the toxicity of the original cationic polymers while retaining their scavenging properties. Together, these data highlight the importance of thromboinflammation in human disease and advance the translational potential of a new class of anti-inflammatory agents.

Stroke is the leading cause of morbidity and the third leading cause of death in the United States. Over 80% of strokes are ischemic in nature, produced by a thrombus occluding the cerebral circulation. Currently, there is only one pharmacologic treatment FDA approved for ischemic stroke; recombinant tissue-type plasminogen activator (rtPA). Unfortunately, thrombolysis with rtPA is underutilized, as it must be administered within three hours of symptom onset and it is not uncommon for treatment to result in intracranial hemorrhage. For these reasons, safe and effective treatments of stroke are a medical necessity.

Aptamers are an attractive emerging class of therapeutic agents that offer additional safety because their activity can be reversed with administration of a complimentary oligonucleotide. Accordingly, I hypothesized that aptamers could be used to treat acute ischemic stroke. First, an antithrombotic aptamer previously generated against coagulation factor IXa was used in a murine model of middle cerebral artery occlusion. Upon factor IXa aptamer administration following stroke, neurological function and inflammatory profiles were improved. Moreover, mice previously treated with the aptamer, followed by induction of subarachnoid hemorrhage, had severe mortality levels and hemorrhage grades that were mitigated by administration of the aptamer's matched antidote.

Second, I generated aptamers against the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1), under the hypothesis that aptamer inhibition of PAI-1 would result in a reversible thrombolytic agent. However, after further testing, the aptamers were not found to disrupt the interaction between PAI-1 and its target proteases. Instead, the aptamers were shown to prevent PAI-1 binding to vitronectin, which translated to restoration of breast cancer cell adhesion in an environment of PAI-1 mediated detachment.

Therefore, aptamer inhibition of factor IXa has demonstrated efficacy in improving outcome following stroke, and should life-threatening hemorrhage arise, an antidote specific to the interventional agent is able to decrease not only hemorrhage grade, but also mortality. This may result in a safer stroke therapy, while a novel aptamer generated against PAI-1 may have application as an antimetastatic agent, which could be used as adjuvant therapy to traditional breast cancer treatment.

Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.

A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.

The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.

To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.

Thrombosis, or pathological blood clot formation, is intimately associated with cardiovascular disease and is the leading cause of morbidity and mortality in the western world. Antithrombotics are commonly prescribed as prophylactic medications or as rapid onset anticoagulants in acute care clinical settings. Although a number of antithrombotics are clinically available, their use is limited by immunogenicity, toxicity, and inability to be controlled with an antidote in the event of hemorrhage. Therefore, new antithrombotics that are effective, yet can be rapidly controlled are urgently needed.

Aptamers are oligonucleotides that form complex secondary and tertiary structures based on intramolecular base pairing and nucleic acid folding that allows them to bind to molecular targets with high affinity and specificity. Aptamers can be isolated that bind to proteins, such as clotting proteins, and modulate protein function. However, unlike most currently used antithrombotics, aptamers can be directly controlled with an antidote and therefore represent a safer class of therapeutic agents.

To generate a novel anticoagulant, we developed an aptamer-antidote pair against prothrombin. Prothrombin is a blood protein that plays an essential role in clot formation. I truncated, optimized, and studied the mechanism of an aptamer that can bind to prothrombin and inhibit prothrombin function, thereby severely impeding clot formation. Moreover, to increase the safety profile of this anticoagulant aptamer, I developed an antidote that can quickly reverse aptamer function and restore normal clotting. This aptamer and antidote pair is the first antidote reversible anticoagulant that targets prothrombin and may prove to be a valuable clinical anticoagulant.

A number of anticoagulants are in development, and a wide debate regarding the optimal protein target for anticoagulation is underway. We have previously generated anticoagulant aptamers to human coagulation factor VII, factor IX, factor X, and prothrombin. I compared the effects of these four anticoagulant aptamers to determine their impact on thrombin generation and clot formation. Each aptamer exerts its own unique effect on thrombin generation/clot formation, depending on the role that its protein target plays in coagulation. These studies provide valuable data regarding target validation and the anticoagulant effects of different therapeutic aptamers.

Robust anticoagulation is required during acute clinical surgical procedures to treat thrombosis. Currently used anticoagulants have several untoward side effects and most are not antidote controllable. I tested the effects of combining two anticoagulant aptamers to assess potential drug synergy. Several combinations of two anticoagulant aptamers were synergistic and severely impaired blood clot formation. One specific pair of aptamers that targeted factor X (FX) and prothrombin in combination was extremely potent and could keep blood fluid in an ex vivo model of extracorporeal circulation. Additionally, this pair of aptamers could be functionally modulated with two different types of antidotes. In conjunction with antidote reversal, this strategy of combining aptamer anticoagulants may prove useful in a variety of highly prothrombotic acute clinical settings.

Finally, to explore the potential of aptamers to regulate platelet function, I isolated and characterized an aptamer toward platelet glycoprotein VI. Glycoprotein VI is a platelet surface receptor that plays a key role in platelet activation and platelet plug formation. I isolated several aptamers that bind to glycoprotein VI, and show that the lead aptamer binds to platelets with high affinity and causes platelet activation and aggregation. This aptamer could potentially be further developed for topical administration to manage bleeding, or for biomarker detection of soluble glycoprotein VI in patient plasma.

In order to ameliorate current maladies, improvements to medicaments and treatment regimens are required. Our lab seeks to translate findings from the laboratory bench to the patient bedside using two approaches: 1) the development of RNA aptamers that bind with high affinity and specificity to defined molecular targets, and 2) repurposing cationic binding polymers as anti-inflammatory agents. This dissertation herein, discusses both of these approaches and summarizes the findings obtained during my graduate training. In the first study, I illustrate how anti-PEG antibodies are capable of binding to and inhibiting a therapeutic RNA aptamer as demonstrated by reduction in drug potency in vitro and in vivo. In the second portion, the development of novel cationic polymer derivatives is discussed, which will help us to determine nucleic acid binding polymer mediated anti-inflammatory mechanisms of action. These findings shed light on the importance of careful and considered drug design to inform the development of future therapeutics. Despite the advances in translational research, there remains a paucity in our understanding of how drugs impact the immune system and this dissertation, in toto, seeks to aid in the development of improved bona fide therapies.

Unfractionated heparin (UFH) is the primary anticoagulant for highly procoagulant indications including cardiopulmonary bypass (CPB) and more recently, severe COVID-19. UFH, however, is unable to fully inhibit thrombin generation leading to continuous activation of both coagulation and inflammatory cascades, increasing the risk for thrombo-inflammatory adverse events. The widespread use of UFH makes it important to improve on its action by addressing its incomplete inhibition of thrombin generation. Additionally, there is a need to better understand how to effectively administer UFH in relation to other therapeutics to avoid counter interactions that leave patients vulnerable to life threatening complications. Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. In the first part of this work, we take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets Factor X/Xa, with UFH (or Low Molecular Weight Heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. In these studies we determined that this aptamer-UFH combination a.) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, b.) allows for a reduced level of UFH to be employed c.) more effectively limits thrombin generation compared to UFH alone and d.) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of Factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation. In the second part of this work, we focused on understanding the interactions between Andexanet alfa (AnXa), a reversal agent for FXa targeted direct oral anticoagulants (DOACs), and UFH. While AnXa was designed to control bleeding associated with the use of DOACs, no studies on its effect on subsequent UFH anticoagulation prior to surgery were carried out. Following FDA approval of AnXa in 2018, several cases have been reported where prior AnXa administration cause heparin resistance during CPB. In our studies, we established that the concomitant presence of AnXa and UFH in whole human blood diminishes the anticoagulant effect of heparin. Furthermore, AnXa addition to ex vivo membrane oxygenation circuit, where UFH anticoagulated blood was being circulated led to the formation of macroscopic and microscopic clots within the circuit. Further studies established potential countermeasures to alleviate heparin resistance in this context through administration of antithrombin (AT) and excess UFH. These results give insight into the interaction of AnXa and UFH during extracorporeal oxygenation and how to circumvent the thrombotic sequalae associated with this interaction. We believe this work will greatly inform and improve patient outcomes. We hope it will also encourage issuance of a warning against concurrent or sequential administration of indirect inhibitors of FXa (UFH, LMWH and Fondaparinux) and AnXa.

Nucleic acids released from dead and dying cells can be recognized as damage-associated molecular patterns (DAMPs) or pattern-associated molecular patterns (PAMPs) by the innate immune system. Unregulated activation of the innate immune response by such endogenous molecules can stimulate pathological inflammation resulting autoimmune disease. Therapeutic efforts have been made to block this inflammation directly by targeting a class of pattern recognition receptors (PRRs), known as Toll-like receptors (TLRs) that recognize such DAMPs and PAMPs, or their downstream signaling molecules. Unfortunately, such therapeutic approaches can suppress immune system and its ability to fight off pathogens. There is a great need for novel therapies that can block this aberrant inflammation prior to TLR and immune recognition, thus allowing normal immune function. A novel class of Nucleic Acid Scavenging Polymers (NASPs) were examined for their ability to do just that. Previously shown to act as nucleic acid scavengers with the ability to neutralize agonists of TLRs NASPs are evaluated for their ability to act prior to TLR activation. Thus, not incurring the non-specific immune suppression evident in other autoimmune therapeutic strategies. Furthermore, NASPs do not limit an animal’s ability to combat viral infection, but rather their administration improves survival when animals are challenged with lethal doses of influenza. The studies outlined in this document suggest that molecules that scavenge extracellular nucleic acids potentially represent promising therapeutic agents to control pathological inflammation associated with a wide range of autoimmune and infectious diseases.

There has been a long history of humans fighting against cancer. Conventional treatments including surgery, radiation therapy, and chemotherapy remain the mainstream approaches, but an increasing understanding of tumor formation and advances in technology have revealed a new approach to cancer treatment: personalized medicine. Personalized medicine considers tumor heterogeneity and tailors treatments to individual patients based on their genetic information and their tumors. Targeted therapy, for example, could precisely attack specific types of cells that express targeted proteins. In recent years, a subclass of targeted therapy, antibody-drug conjugates (ADC), has received vast clinical attention due to their ability to deliver highly toxic drugs to cancer cells and effectively kill them while sparing healthy cells. Intrigued by the working philosophy of ADCs while acknowledging their limitations, a group of scientists, including our lab, proposed the use of aptamers to create a new class of targeted therapeutics. Aptamers are RNA or DNA ligands that do not require humanization and pose minimal immunogenic risks to patients. Previously, our group reported an RNA aptamer, named E3, which was selected to target prostate cancer cells and demonstrated the ability to effectively eliminate cancer cells when conjugated with drugs. Here, I observe that E3 can also target a broad range of other cancer types, leading me to investigate its molecular target. The following study shows that the E3 aptamer targets human transferrin receptor 1 (hTfR) to enter the cancer cells, consistent with the upregulated expression of hTfR in most cancer types. However, I encountered challenges in the next-step laboratory development of E3 since it did not exhibit cross-reactivity in murine cells. Therefore, I demonstrated that E3 also targets canine cancer cells, which highlights the potential to test E3 in canine models. To further develop a TfR targeting aptamer that can work in both mouse models and against human cancer, I performed a new selection for an aptamer using 2’OMe A, C, U, and 2’OH G modified RNA library that can recognize both human and murine TfR with better resistance for nuclease degradation. Additionally, a non-transferrin (Tf) competing TfR-aptamer is also identified in the study with more nuclease resistance. The results of this study offer several potential weapons used for treating cancers. E3 aptamer targeting hTfR works well in human cancer xenograft mouse models but encounters challenges for characterizations in vivo. Therefore, I select and report a panel of TfR-targeting aptamers that can be used for mouse study or clinical development. Through this study, I aim to contribute to the advancement of targeted delivery and improve drug efficacy in cancer patients.

Thrombosis is associated with the occlusion of a blood vessel and can be triggered by a number of types of injury, such as the rupture of an atherosclerotic plaque on the artery wall, changes in blood composition, or blood stasis. The resulting thrombosis can cause major diseases such as myocardial infarction, stroke, and venous thromboembolic disorders that, collectively, account for the most common cause of death in the developed world. Anticoagulants are used to treat and prevent these thrombotic diseases in a number of clinical and surgical settings. Although commonly prescribed, currently approved anticoagulants have a major limitation of severe drug-induced bleeding that contributes to the high levels of morbidity and mortality associated with use. The "holy grail" for antithrombotic therapy is to identify a drug that inhibits thrombus formation without promoting bleeding. Understanding the differences between thrombosis and hemostasis in the vascular system is critical to developing these safe and effective anticoagulants, as this depends on striking the correct balance between inhibiting thrombus formation (efficacy) and reducing the risk of severe bleeding (safety). While it is commonly thought that the same factors play a similar role in hemostasis and thrombosis, recent evidence points to differing functions for FXI and FXII in each of these settings. Importantly, these factors seem to contribute to pathological thrombus formation without being involved in normal hemostasis.

The overall goal of this project was to evaluate the inhibition of the intrinsic pathway of coagulation as a potential anticoagulant strategy utilizing the aptamer platform. Aptamers are short, highly structured nucleic acids that act as antagonists by binding to large surface areas on their target protein and thus tend to inhibit protein-protein interactions. High affinity binding aptamers have been isolated that specifically target a diverse range of proteins, including transcription factors, proteases, viral proteins, and growth factors, as well as other coagulation factors. As synthetic molecules, aptamers have a small molecular weight, are highly amenable to modifications that can control their bioavailability, and have not been found to elicit an immune response, thus making them ideal drug candidates. Importantly, aptamers can be rapidly and effectively reversed with either a sequence specific antidote that recognizes the primary sequence of the aptamer or a universal antidote that binds to their backbone and reverses all aptamer activity independent of sequence. This ability lends itself well to their therapeutic application in coagulation, as rapid reversal of a drug upon the onset of bleeding is a key property for increasing the safety of this class of drugs.

Aptamers targeting FXI/FXIa and FXII/FXIIa were isolated in two separate SELEX (systematic evolution of ligands by exponential enrichment) procedures: the FXII aptamer was isolated in a convergent SELEX approach and the FXIa aptamer was isolated from a purified protein selection. In both processes, 2'fluoropyrimindine modified RNA with a 40-nucleotide random region was incubated with either the plasma proteome (in initial rounds of the convergent SELEX) or the purified protein target (FXII or FXIa). The nucleic acids that did not bind to the target were separated from those that bound, and these molecules were then amplified to generate an enriched pool with increased binding affinity for the target. This process was repeated under increasingly stringent conditions to isolate the aptamer that bound with the highest affinity to the purified target protein. Utilizing biochemical and in vitro coagulation assays, specific, high-affinity binding and functional anticoagulant aptamers were identified for both protein targets, and the mechanism of anticoagulation was ascertained for each aptamer.

Overall, both aptamers bound to an exosite on their target protein that was able to inhibit downstream activation of the next protein in the coagulation cascade. In order to specifically examine aptamer effects on several parameters of thrombin generation, a new assay was developed and fully characterized using aptamer anticoagulants targeting other coagulation factors. Aptamer inhibition of both FXI and FXII was able to decrease thrombin generation in human plasma. However, limited cross-reactivity in other animal species by both aptamers hindered our ability to assess aptamer inhibition in an in vivo setting. Moving forward, screening aptamers against a larger selection of animal plasmas will hopefully allow us to identify an animal species in which we can analyze aptamer inhibition of the intrinsic pathway for effectiveness and safety in inhibiting thrombosis. The further characterization and use of these aptamers in plasma and blood based settings will allow us to study the diverging functions of the intrinsic pathway in thrombosis and hemostasis.

A critical need exists for safe and effective anticoagulants to treat and prevent numerous thrombotic procedures and diseases. An ideal anticoagulant is one that strikes the correct balance between inhibiting thrombus formation and reducing drug-induced bleeding. Inhibition or depletion of factors XI and XII of the intrinsic pathway of coagulation have shown reduced thrombus formation without interruption of normal hemostasis in several models of thrombosis. By developing novel RNA aptamer anticoagulants to these factors, we have set the stage for evaluating the net therapeutic benefit of intrinsic pathway inhibition to effectively control coagulation, manage thrombosis, and improve patient outcome. As well as developing a safe anticoagulation, these agents can lead to important biological discoveries concerning the fundamental difference between hemostasis and thrombosis.

While much of the current focus on advancing CRISPR-Cas9 editing revolves around the engineering of Cas9, the interrogation and evolution of sgRNA scaffold, in addition to novel Cas9 binding RNAs, represent another echelon of development and therapeutic potential. Currently, the majority of research utilizes a singular guide RNA scaffold sequence (the sgRNA) for a given Cas protein (e.g., the Streptococcus pyogenes Cas9 and associated guide RNA). This sequence inflexibility makes many sites within the genome intractable to CRISPR/Cas editing, often due to undesirable intramolecular interactions that result in undesirable secondary structures. Additionally, given the electrostatic potential of Cas9, it may be possible to generate additional Cas9 binding RNA molecules.Here, we use utilize SELEX to both identify novel Cas9 binding RNAs and interrogate the sequence depth of the sgRNA scaffold. First, a SELEX scheme utilizing a nitrocellulose filter binding assay was utilized to identify modified RNA aptamers that bind to Cas9 with specificity and an affinity rivaling that of the sgRNA. The aptamer was shown to tolerate truncations and sequence additions, demonstrating an ability to localize oligonucleotide-based therapeutics to the Cas9 protein. We believe that this aptamer can be expanded upon to incorporate novel functions instead of altering the sgRNA . Second, we use a novel combinatorial approach that utilizes a functional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify numerous, diverse sgRNA variants that bind S. pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in the sgRNA sequence and are utilized in a combinatorial approach to identify scaffolds that enhance editing efficiencies when paired DNA-binding antisense domains. Using molecular evolution, guide RNA scaffolds can be generated for specific targets and optimized to ensure that secondary structure is maintained. This selection approach should be valuable for generating gRNAs with a range of new activities.

Breast cancers (BC) remain the most lethal malignancies amongst women. Subtype heterogeneity and aggressive invasion are believed to be major contributors for poor outcomes. Triple negative breast cancers (TNBC) are notoriously aggressive, difficult to treat, pro-inflammatory and highly metastatic. Patients that are diagnosed with localized, surgically resectable tumors are treated with neoadjuvant (preoperative) and adjuvant (post-operative) chemotherapy in the hopes of eliminating micro- and oligo-metastatic disease. However, a majority of these patients progress to metastatic disease even under the selective pressure of our current aggressive therapeutic armament. Tumor metastases to tropic organ sites – primarily the lungs, bones, and brain – can wreak havoc on patients with triple-negative breast cancer. Unfortunately, most patients with metastatic TNBC decline quickly. In order to change the tide against pancreatic cancer and other aggressively metastatic tumor types, there need to be new clinical approaches in the arena of anti-metastatic therapeutics.Tumor metastasis is an incredibly complex process involving interactions between tumor cells, the tumor microenvironment (TME), the surrounding stromal tissues, blood and lymphatic vessels, and the innate and adaptive immune systems. The earliest steps of the metastatic cascade require the tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) in order to have the appropriate morphology, detach from the primary tumor site, invade/intravasate into the stroma and reach the host’s blood or lymphatic vessels in order to travel to distant sites. Even once in the circulation, the tumor cell has to survive the turbulent hemodynamic environment before embedding itself into the distant organ site. Various immune sensors also contribute to this complex interplay, especially the toll-like family of receptors (TLRs). These receptors evolved as a part of the innate immune system to detect pathogen-associated molecular patterns (PAMPs) to combat infectious pathogens including bacteria, fungi and viruses. They can also respond to molecular motifs from self – also known as damage-associated molecular patterns (DAMPs). These receptors are typically expressed by immune cells such as macrophages, dendritic cells, and neutrophils but in recent times have been shown to be expressed on both cancer cells and stromal cells within the tumor. The expression and activation of these receptors by either PAMPs and/or DAMPs has been implicated in tumor progression and metastasis in a variety of solid and hematologic malignancies. Going beyond their utility to act as universal antidotes for aptamers (which was already beyond their initial purpose as gene delivery tools), our lab previously discovered that a subset of nucleic-acid binding polymers (NABPs) could behave as nucleic -acid scavengers (NAS). Previously we determined that NASs can block metastatic signals elicited by nucleic acid-containing damage-associated molecular patterns (NA DAMPs) in the pancreatic cancer setting. By behaving as anti-inflammatory compounds scavenging extracellular nucleic acids and associated protein complexes that promote pathological activation of TLRs, our lab demonstrated their efficacy in various murine models of disease including systemic lupus erythematosus, sepsis, and influenza infection. Nucleic-acid mediated TLR signaling also facilitates tumor progression and metastasis in several malignancies, including pancreatic cancer and breast cancer. In the first part of this dissertation, we observe that TNBC cells express TLR9, are responsive to TLR9 ligands, and treatment of TNBC cells with chemotherapy increases the release of such NA DAMPs in culture. Chemotherapy derived and BC-patient derived DAMPs increase TLR9 activation and TNBC cell invasion in vitro; however, treatment with the NAS PAMAM-G3 significantly counteracts such effects. NAS treatment in a spontaneous BC murine model (MMTV-PyMT) also led to diminished lung metastatic burden. Thus, NAS may prove useful for inhibiting pathological processes (e.g. metastasis) and represent a novel combination therapeutic approach. The second part of this dissertation, we focused on a clinical descriptive project whereby we took plasma from breast cancer patients pre- and post-neoadjuvant chemotherapy. Our goal was to get an overview of the immune landscape in these patients and if there was any functional consequence of chemotherapy treatment on invasive potential in vitro. While the work shows promise, it really is laying down the groundwork for better understanding what immune mediators are upregulated upon standard-of-care therapies. The last part of this dissertation covers ongoing projects that I started but was unable to bring all the way to publication. Among these are the optimization of cytokine arrays using monocytes and various sources of in vitro or ex vivo NA DAMPs. I also detail a little of the work we carried out building the immune panels to better track and interrogate antigen-tagged pancreatic cancer cells and where they metastasize as well as the effect of PAMAM-G3 in our murine models of cancer. And finally, a small vignette concerning the role of cancer-associated tumor thrombosis and how we can utilize the highly pro-thrombotic KPC pancreatic cancer mouse model to be imaged via a fluorescence labeled thrombin aptamer.

Pancreatic cancer is a devastating disease with the worst prognosis of all the major cancers with a 5-year survival of less than 7%. This is due, in part, to the insidious development of pancreatic cancer which is largely asymptomatic until the tumor is too far developed to be efficiently combatted. The poor prognosis of pancreatic cancer is also due to its aggressively metastatic nature. Patients that are diagnosed with localized, surgically resectable tumors are treated with neoadjuvant (preoperative) and adjuvant (post-operative) chemotherapy, with or without radiation therapy, in the hopes of eviscerating micrometastatic disease. However, many of these patients progress to metastatic disease even in the face of aggressive therapeutic intervention. Tumor metastases to other organ sites, primarily the liver, wreak havoc on patients with pancreatic cancer. Unfortunately, most patients with metastatic pancreatic cancer decline quickly. In order to change the tide against pancreatic cancer and other aggressively metastatic tumor types, there need to be new clinical approaches in the arena of anti-metastatic therapeutics.

Tumor metastasis is an incredibly complex process involving interactions between tumor cells, the tumor microenvironment, the peripheral stromal tissues, hematologic and lymphatic vessels, and the immune system. In order to reach a metastatic site, the tumor cell has to undergo epithelial to mesenchymal transition, detaching itself from its primary site, traversing the tumor stroma and extracellular matrix to reach hematologic or lymphatic vessels, traveling through the vasculature, and then embedding itself into the distant organ site. Contributing to the complexity of tumor metastasis are the toll-like family of receptors (TLRs). These receptors evolved as a part of the innate immune system to detect pathogen associated molecular patterns (PAMPs) to combat infectious agents such as bacteria and viruses. They are also known, however, to respond to molecules released by innate cells known as damage associated molecular patterns (DAMPs). These receptors are typically expressed by immune cells such as dendritic cells or neutrophils but have more recently been discovered to be expressed by both cancer cells and stromal cells within the tumor. The expression and activation of these receptors by both PAMPs and DAMPs has been implicated in tumor progression and metastasis in a multitude of cancers.

Extracellular nucleic acids and nucleic acid-protein complexes have come to the fore as DAMPs that activate nucleic acid sensing TLRs such as TLR9 and TLR4, which sense DNA and nucleosomes, respectively. These innate molecules and their recognition receptors have been strongly implicated in tumor progression and metastasis in lung, breast, skin and pancreatic cancers. Studies have shown that specific activation of TLRs 4 and 9 contribute to invasion in vitro and metastasis in vivo. Recent work has also shown that DNAse treatment in animal models of cancer reduces metastatic burden. Thus, targeting extracellular nucleic acids and nucleic acid-protein complexes seems like a therapeutic strategy that warrants further exploration.

In addition to circulating freely, nucleic acids and nucleic acid-protein complexes can also circulate in tumor derived lipid particles such as microparticles and exosomes. Recent research has shown these particles to have a myriad of activities including autocrine signaling amongst tumor cells, paracrine signaling between tumor cells, and communication with tissues of distant metastatic sites. Specifically, studies have shown that tumor derived microparticles can induce invasion in breast cancer cells. Additionally, pancreatic cancer derived exosomes have been shown to pre-condition the liver for metastatic implantation. The discovery of the diverse activities these particles are capable of has uncovered a new layer of the complexity via which tumors communicate and metastasize. These discoveries have also yielded new therapeutic targets that may yield novel clinical approaches to limit tumor progression and metastasis.

Cationic polymers were initially developed exclusively as tools for gene delivery. Our group has previously shown that these polymers have the ability to bind to nucleic acid and nucleic-acid protein complexes and neutralize their ability to activate their specific TLRs. This anti-inflammatory ability of cationic polymers has led to beneficial effects in multiple mouse models of diseases in which nucleic acid mediated TLR signaling and inflammation are essential to disease progression. For example, in mouse models of acute liver failure, cutaneous lupus erythematosus, and influenza, cationic polymer therapy decreased the severity of the inflammatory response and increased survival in treated mice. These studies gave light to the new possibilities never before considered of using cationic polymers as multifaceted neutralizing agents of extracellular nucleic acid mediated inflammation.

It is after considering all of the previous observations in aggregate that the work described in this thesis was undertaken. Pancreatic cancer was chosen as the disease model due to its aggressively metastatic nature. We first quantified the levels of extracellular nucleic acids and nucleic acid-protein complexes from the sera of patients with known pancreatic cancer. We found that the sera of patients with early stage pancreatic cancer had elevated levels of cell-free DNA (cfDNA), nucleosomes, and HMGB-1 as compared to sera from healthy volunteers. We also found that these factors increased in the sera of pancreatic cancer patients with standard therapies such as combination chemo-radiation therapy and surgical resection of the tumor. After quantifying these circulating factors, the next step was to interrogate the functional significance of elevated cfDNA, nucleosomes, and HMGB-1. Using a panel of TLR reporter cell lines, we tested the ability of pancreatic cancer serum to activate TLRs. Interestingly these receptors were strongly activated by pancreatic cancer patient sera and this activity was neutralized in some TLRs by cationic polymer treatment.

TLR expression by cancer cells has been observed in the literature and activation of these receptors on tumor cells has been shown to induce invasive and aggressive phenotypes. We confirmed that a number of pancreatic cancer cell lines express TLRs and numerous studies from the literature show that tumor sections from patients with pancreatic cancer show increased staining for TLRs 4 and 9. Next we investigated the functional significance of TLR expression in pancreatic cancer cell lines using a Transwell-Matrigel invasion assay. We observed that treatment with DNA based TLR agonists induced a significantly invasive phenotype in a multitude of pancreatic cancer cell lines. This pro-invasive effect was abrogated with cationic polymer treatment. Next, we directly tested the pancreatic cancer patient serum in our invasion assay to ascertain whether the TLR9 activity we observed in our TLR reporter cell assays translated to functional effects on tumor cells. Indeed, pancreatic patient sera induced significant invasion in a Transwell-Matrigel invasion assay when compared to sera from healthy volunteers. Moreover, cationic polymer treatment significantly inhibited the ability of cancer patient sera to induce invasion.

In addition to extracellular nucleic acids and nucleic acid-protein complexes, microvesicles derived from cultured tumor cells were also tested for their ability to induce invasion in the Transwell-Matrigel invasion assay. Interestingly, both microparticles and exosomes isolated from cultured tumor cells induced invasion in tumor cells and concurrent cationic polymer treatment significantly inhibited the pro-invasive phenotype. This observation provided a basis for the multi-modal use of cationic polymers as potential inhibitors of tumor invasion.

In order to elucidate the use of cationic polymers as cancer therapeutics, we established a syngeneic, bioluminescent, immunocompetent murine model of pancreatic cancer metastasis. This model facilitated our ability to track the progress of liver metastases as well as growth of the primary tumor in an immunocompetent setting, thus allowing a more accurate recapitulation of tumor growth and metastasis as it occurs in humans. Cationic polymer therapy significantly reduced liver metastases in our model of pancreatic cancer. Specifically, liver metastases were greatly reduced as measured by total bioluminescent flux (tumor cell specific) and by gross organ weight. In fact, liver weights in polymer treated mice were equivalent to weights from the livers of healthy mice. Liver metastases in polymer treated mice were reduced by >90% overall. Interestingly, polymer treatment had no effect on primary tumor growth as measured by bioluminescent flux and gross organ weight. The lack of any effect on the primary tumor is supported by the in vitro data that we gathered, showing that cationic polymer treatment inhibited tumor invasion but had no effect on tumor cell viability.

The second portion of this thesis was focused on characterizing the nucleic acids and nucleic acid-protein complexes in the cancer patient sera that were bound to the cationic polymers. In order to accomplish this goal, we chemically modified an existing cationic polymer to add a biotin tag to the polymer, allowing us to pull the polymer and bound molecules out of solutions using streptavidin coated beads. By performing this modification, we were able to take cancer patient sera and pullout the polymer-nucleic acid/protein complex. By doing so, we were able to show that A) pulling the bound species out of the sera reduced the ability of the sera to activate TLRs and B) The bound species that is pulled out of the sera is indeed responsible for the activation of TLRs.

In order to be able to do more in depth analysis of the molecules being bound by the polymers, we had to increase the scale of our source material beyond patient serum due to the limited nature of harvesting blood from patients with pancreatic cancer. To accomplish this, we cultured human pancreatic cell lines to confluence and collected conditioned media from these cells. Cells with media alone represented patients with untreated pancreatic cancer. Cells were also treated with radiation prior to collection of conditioned media to recapitulate the fractionated radiation patients with pancreatic cancer receive. This gave us a resource for biomolecules that would represent the secretions of pancreatic tumors under a variety of conditions.

The next step was to characterize the polymer bound molecules, analyzing the nucleic acid and proteinaceous components pulled out of the conditioned media. In order to accomplish this analysis, we displaced the polymer bound molecules using soluble heparin, a negatively charged polymer that would knock off negatively charged DNA and DNA-protein complexes. Using this strategy, we were able to isolate the proteinaceous and nucleic acid components of the polymer bound molecules. The protein components were sent for qualitative mass spectrometry which yielded numerous known markers of pancreatic cancer and agonists of TLR4. The nucleic acid components of the polymer bound molecules were run on agarose gels to analyze their size patterns. The gels yielded a wide diversity of bands, representing the variety of DNA sizes that are released by cancer cells at baseline and in response to radiation therapy.

In addition, we adopted a parallel strategy to characterize the proteins the polymer was binding to by analyzing the conditioned media before and after biotinylated-polymer treatment with ELISA’s specific for HMGB-1 and nucleosomes. These proteins are important mediators of TLR based inflammation, particularly due to their ability to bind to and act as carriers for pro-inflammatory nucleic acids that potently activate TLR9. Using this strategy, we discovered that after pulling out the biotinylated polymer, the concentrations of HMGB-1 and nucleosomes decreased in the conditioned media. Additionally, both protein complexes were found in the heparin displaced supernatant from the polymer. The polymer treated and untreated conditioned media were incubated with our TLR reporter cells and we observed decreased TLR activity in the conditioned media that was treated with biotinylated polymer. This strategy also confirmed our previous observation that in addition to nucleic acids, the polymer is binding to nucleic acid-protein complexes and preventing them from activating TLRs. This strategy was also repeated with different cell types, including immortalized, normal pancreatic epithelial cells, human endothelial cells, and lymphoid cells to encompass the cell types that are likely exposed during standard radiation therapy. Interestingly, the most potent release of TLR agonists was seen from tumor cells with other cell types having some TLR activity but significantly less than tumor cells.

The use of polycationic polymers as anti-metastatic agents in cancer has never before been considered as a potential use for these incredibly malleable tools. Moreover, the ability of these polymers to be chemically modified allows their use as potential therapeutic agents, tools for liquid biopsy of patient derived and cell culture samples, and a means for understanding the mechanistic underpinnings of the in vivo efficacy of polymers in cancer models. The work described here highlights the diverse capabilities of polycationic polymers in pancreatic cancer. They act as potent anti-inflammatory agents, reducing the levels of TLR activation. This activity translates to an inhibition of in vitro invasion in pancreatic cancer cell lines which is further reflected in the significant inhibition of liver metastasis in vivo. The potential clinical benefits are further explained with the use of a biotin-modified polymer to bind, extract, and identify the pro-inflammatory nucleic acids and nucleic-acid protein complexes. The use of this modified polymer yielded both the identities of the bioactive molecules as well as provide a means to perform a liquid biopsy of cell culture media and patient sera. In totality, the work described here signifies the diverse capability of polycationic polymers in cancer.