Browsing by Subject "Aptamer"

Creation of novel tools for biomedical applications is critical for the improvement of patient diagnostics and therapeutics. Two particularly important needs lie in (1) improved in vitro testing and increased performance of prosthetic vascular grafts and (2) purification methods for cells that do not compromise their utility. Progress in these areas is urgently needed and would facilitate the availability of higher quality devices and treatments that raise the quality of patient care. This work focused on developing new approaches toward that goal.

A tremendous and immediate need exists for high-performance small-diameter synthetic vascular grafts, as a fifth of the 500,000 annual coronary artery bypass grafting (CABG) patients lack suitable autologous vessels for revascularization. This problem has driven intense research and development of increasingly diverse prosthetics that could be viable alternatives in the years to come. Evaluating these designs in vitro offers high-throughput, low-cost screening for promising graft technologies ahead of more stringent vetting in vivo.

Offering a fresh take on assessing vascular graft thrombogenicity in vitro, the buildup of pressure upstream to a clot was used as a metric to quantify the physical interaction between the graft lumen and a maturing thrombus. A closed tubing system was devised and continuously monitored as clotting solutions of fibrin glue, platelet-rich plasma or whole blood were cured to varying maturities and then purged from small-diameter ePTFE grafts or Tygon graft mimics. This approach provided insight into how blood flow resistance is influenced by a number of clinically relevant factors, such as the level of vessel occlusion and the physical nature of the resident coagulum.

Endothelialization of synthetic vascular grafts yields viable alternatives to native vessels and can be accomplished non-invasively using late-outgrowth endothelial progenitor cells (LO-EPCs) isolated from peripheral blood. However, the time required to amass sufficient cells to prepare a graft with current methods is risky for waiting CABG patients. An ambitious approach conceived to significantly decrease this wait period involved developing affinity ligands selective for LO-EPCs that would enable their capture directly from the circulation to facilitate rapid amassment. An in vitro directed evolution strategy to generate aptamers, the nucleic acid analogs of antibodies, that specifically bind these cells was carried out with initially promising but ultimately unsuccessful results. While the particular strategy executed here did not prevail, the high value and impact LO-EPC aptamers would deliver merit revisiting this work with a revised strategy such as the one proposed in this document.

Purification of autologous and allogenic cells is essential for their use in a variety of therapeutic and basic research applications in addition to augmenting graft performance. However, the antibody stains conventionally used to selectively purify cells are permanent and their continued presence can elicit an immune response in vivo and compromise native cell behavior. To avoid these issues, a cell purification strategy was crafted utilizing aptamers and matched oligonucleotide antidotes that enabled reversible cell staining. The reversible stains were robust enough for cell purification via fluorescence-activated cell sorting (FACS) yet subsequently able to be removed with gentle heat treatment and antidote. Importantly, cell function that was compromised without antidote was rescued to match the native behavior of non-stained cells following purification and antidote treatment.

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.

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.

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.

Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities in the millions that suffer TBI every year. Important mediators of inflammation include macrophages and microglia and these cell types can possess a range of phenotypes. An anti-inflammatory, “M2-like” macrophage phenotype after TBI is associated with neurogenesis, axonal regeneration, and improved white matter integrity. To boost these subpopulations, a promising approach is the enrichment of two cytokines: Fractalkine (FKN, CX3CL1) or Interleukin-4 (IL-4). FKN is a chemokine and thus recruits non-classical monocytes which are precursors to M2-like macrophages. IL-4 polarizes and proliferates M2-like macrophages. However, delivering recombinant or purified cytokines is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we explore two strategies to enrich endogenous FKN or IL-4, obviating the need for delivery of exogenous proteins.

In the first study, we synthesize a biomaterial to elevate endogenous FKN at an injury site. Modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels (“aptagels”) that dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate local increases in non-classical monocytes and M2-like macrophages.

In our second approach, we augment mesenchymal stem/stromal cells (MSCs), to transiently express IL-4. As MSCs do not endogenously synthesize IL-4, we transfect them with synthetic IL-4 mRNA. We suggest that mRNA transfection is a better strategy than DNA transfection, viral transduction, and recombinant IL-4 delivery for TBI. Our studies first characterize the IL-4 expression. Then, in a TBI model of closed head injury, we observe that IL-4 expressing MSCs successfully induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression. Curiously, this does not translate to improvements in function, histology, or white matter integrity.

The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins. Both FKN-aptamer functionalized biomaterials and IL-4 expressing MSCs may be promising approaches to boost anti-inflammatory subpopulations at sites of injury. However, our studies also begin to question whether M2-like macrophages alone orchestrate the neurogenesis, axonal regeneration, and improved white matter integrity that has previously been observed.

Finally, both strategies could have important immunomodulatory roles outside of TBI. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic. IL-4 expressing MSCs could assist tissue regeneration in cavitary diseases or improve biomaterial integration into tissues.

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.

In addition to being the natural genetic information carrier, DNA can also serve as a versatile material for construction of nanoscale objects. By using the base-pairing properties of DNA, we have been able to mass-produce nano-scale structures in a variety of different shapes, upon which patterns of other molecules can be further specified. The diversity of molecules and materials that can be attached to DNA and the capability of providing precise spatial positioning considerably enhance the attractiveness of DNA for nano-scale construction. A further challenge remains to use these DNA based structures for biomedical applications.

As proof-of-concept, a DNA-based nanodevice for multivalent thrombolytic delivery is designed, which intends to employ DNA nanostructures as carriers for the delivery of tissue plasminogen activator (tPA) and plasminogen. Universal modular adapter molecules that can simultaneously bind "down" to the DNA structures and "up" to these thrombolytic drugs are further proposed. We begin with exploring the molecular recognition properties provided by biotin-avidin and aptamer-ligand pairs, and are able to achieve site-specific display of certain protein targets along the DNA nanostructure scaffold. Yet for both of these approaches, only biotinylated or specially selected proteins can be patterned. We further propose to develop single-chain diabodies (scDb) as the adapter molecules. This scDb approach is highly modular and can be extended to assemble virtually any proteins and therapeutic molecules of interests, which at the same time will greatly enhance our molecular toolbox for nanoscale construction.

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.