CaMKII (Ca2+/Calmodulin-Dependent Kinase II) in Mitochondria of Smooth Muscle Cells Controls Mitochondrial Mobility, Migration, and Neointima Formation.

OBJECTIVE: The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS: The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS: These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.

AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization.

Analyzing HT-SELEX data with the Galaxy Project tools--A web based bioinformatics platform for biomedical research.

The development of DNA and RNA aptamers for research as well as diagnostic and therapeutic applications is a rapidly growing field. In the past decade, the process of identifying aptamers has been revolutionized with the advent of high-throughput sequencing (HTS). However, bioinformatics tools that enable the average molecular biologist to analyze these large datasets and expedite the identification of candidate aptamer sequences have been lagging behind the HTS revolution. The Galaxy Project was developed in order to efficiently analyze genome, exome, and transcriptome HTS data, and we have now applied these tools to aptamer HTS data. The Galaxy Project's public webserver is an open source collection of bioinformatics tools that are powerful, flexible, dynamic, and user friendly. The online nature of the Galaxy webserver and its graphical interface allow users to analyze HTS data without compiling code or installing multiple programs. Herein we describe how tools within the Galaxy webserver can be adapted to pre-process, compile, filter and analyze aptamer HTS data from multiple rounds of selection.

Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-ß phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease.

Phosphorylation of Nox1 regulates association with NoxA1 activation domain.

RATIONALE: Activation of Nox1 initiates redox-dependent signaling events crucial in the pathogenesis of vascular disease. Selective targeting of Nox1 is an attractive potential therapy, but requires a better understanding of the molecular modifications controlling its activation. OBJECTIVE: To determine whether posttranslational modifications of Nox1 regulate its activity in vascular cells. METHODS AND RESULTS: We first found evidence that Nox1 is phosphorylated in multiple models of vascular disease. Next, studies using mass spectroscopy and a pharmacological inhibitor demonstrated that protein kinase C-beta1 mediates phosphorylation of Nox1 in response to tumor necrosis factor-α. siRNA-mediated silencing of protein kinase C-beta1 abolished tumor necrosis factor-α-mediated reactive oxygen species production and vascular smooth muscle cell migration. Site-directed mutagenesis and isothermal titration calorimetry indicated that protein kinase C-beta1 phosphorylates Nox1 at threonine 429. Moreover, Nox1 threonine 429 phosphorylation facilitated the association of Nox1 with the NoxA1 activation domain and was necessary for NADPH oxidase complex assembly, reactive oxygen species production, and vascular smooth muscle cell migration. CONCLUSIONS: We conclude that protein kinase C-beta1 phosphorylation of threonine 429 regulates activation of Nox1 NADPH oxidase.

Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers.

Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

AptamerRunner: An accessible aptamer structure prediction and clustering algorithm for visualization of selected aptamers.

Aptamers are short single-stranded DNA or RNA molecules with high affinity and specificity for targets and are generated using the iterative Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. Next-generation sequencing (NGS) revolutionized aptamer selections by allowing a more comprehensive analysis of SELEX-enriched aptamers as compared to Sanger sequencing. The current challenge with aptamer NGS datasets is identifying a diverse cohort of candidate aptamers with the highest likelihood of successful experimental validation. Herein we present AptamerRunner, an aptamer clustering algorithm that generates visual networks of aptamers that are related by sequence and/or structure. These networks can then be overlayed with ranking data, such as fold enrichment or data from scoring algorithms. The ability to visually integrate data using AptamerRunner represents a significant advancement over existing clustering tools by providing a natural context to depict groups of aptamers from which ranked or scored candidates can be chosen for experimental validation. The inherent flexibility, user-friendly design, and prospects for future enhancements with AptamerRunner has broad-reaching implications for aptamer researchers across a wide range of disciplines.

Selection of a novel cell-internalizing RNA aptamer specific for CD22 antigen in B cell acute lymphoblastic leukemia.

Despite improvements in B cell acute lymphoblastic leukemia (B-ALL) treatment, a significant number of patients experience relapse of the disease, resulting in poor prognosis and high mortality. One of the drawbacks of current B-ALL treatments is the high toxicity associated with the non-specificity of chemotherapeutic drugs. Targeted therapy is an appealing strategy to treat B-ALL to mitigate these toxic off-target effects. One such target is the B cell surface protein CD22. The restricted expression of CD22 on the B-cell lineage and its ligand-induced internalizing properties make it an attractive target in cases of B cell malignancies. To target B-ALL and the CD22 protein, we performed cell internalization SELEX (Systematic Evolution of Ligands by EXponential enrichment) followed by molecular docking to identify internalizing aptamers specific for B-ALL cells that bind the CD22 cell-surface receptor. We identified two RNA aptamers, B-ALL1 and B-ALL2, that target human malignant B cells, with B-ALL1 the first documented RNA aptamer interacting with the CD22 antigen. These B-ALL-specific aptamers represent an important first step toward developing novel targeted therapies for B cell malignancy treatments.

Combination of protein and cell internalization SELEX identifies a potential RNA therapeutic and delivery platform to treat EphA2-expressing tumors.

The EphA2 receptor tyrosine kinase is overexpressed in most solid tumors and acts as the major driver of tumorigenesis. In this study, we developed a novel approach for targeting the EphA2 receptor using a 2'-fluoro-modified pyrimidine RNA aptamer termed ATOP. We identified the ATOP EphA2 aptamer using a novel bioinformatics strategy that compared aptamers enriched during a protein SELEX using recombinant human EphA2 and a cell-internalization SELEX using EphA2-expressing MDA231 tumor cells. When applied to EphA2-expressing tumor cell lines, the ATOP EphA2 aptamer attenuated tumor cell migration and clonogenicity. In a mouse model of spontaneous metastasis, the ATOP EphA2 aptamer slowed primary tumor growth and significantly reduced the number of lung metastases. The EphA2 ATOP aptamer represents a promising candidate for the development of next-generation targeted therapies that provide safer and more effective treatment of EphA2-overexpressing tumors.

Optimization of RNA Aptamer SELEX Methods: Improved Aptamer Transcript 3'-End Homogeneity, PAGE Purification Yield, and Target-Bound Aptamer RNA Recovery.

Since its inception in the early 1990s, SELEX remains the gold standard for discovering RNA aptamers specific for proteins and small molecules. The SELEX process has undergone countless modifications and now encompasses a breadth of innovative selection schemes to pare an aptamer library toward target-specific aptamers. Common to all these RNA aptamer SELEX processes are the steps for the preparation of DNA template and in vitro transcription of aptamer RNA. These steps have remained mostly unchanged over the past three decades and would benefit from optimization. We focused on three key areas: improving the homogeneity of in vitro transcribed aptamer RNA, increasing the efficiency of in vitro transcribed aptamer RNA purification by PAGE, and improving the quality of target-bound aptamer RNA recovered during SELEX. Together, these optimizations contribute toward a more efficient SELEX process and are applicable to both protein-based and cell-based RNA aptamer selections.

Proarrhythmic defects in Timothy syndrome require calmodulin kinase II.

BACKGROUND: Timothy syndrome (TS) is a disease of excessive cellular Ca(2+) entry and life-threatening arrhythmias caused by a mutation in the primary cardiac L-type Ca(2+) channel (Ca(V)1.2). The TS mutation causes loss of normal voltage-dependent inactivation of Ca(V)1.2 current (I(Ca)). During cellular Ca(2+) overload, the calmodulin-dependent protein kinase II (CaMKII) causes arrhythmias. We hypothesized that CaMKII is a part of the proarrhythmic mechanism in TS. METHODS AND RESULTS: We developed an adult rat ventricular myocyte model of TS (G406R) by lentivirus-mediated transfer of wild-type and TS Ca(V)1.2. The exogenous Ca(V)1.2 contained a mutation (T1066Y) conferring dihydropyridine resistance, so we could silence endogenous Ca(V)1.2 with nifedipine and maintain peak I(Ca) at control levels in infected cells. TS Ca(V)1.2-infected ventricular myocytes exhibited the signature voltage-dependent inactivation loss under Ca(2+) buffering conditions, not permissive for CaMKII activation. In physiological Ca(2+) solutions, TS Ca(V)1.2-expressing ventricular myocytes exhibited increased CaMKII activity and a proarrhythmic phenotype that included action potential prolongation, increased I(Ca) facilitation, and afterdepolarizations. Intracellular dialysis of a CaMKII inhibitory peptide, but not a control peptide, reversed increases in I(Ca) facilitation, normalized the action potential, and prevented afterdepolarizations. We developed a revised mathematical model that accounts for CaMKII-dependent and CaMKII-independent effects of the TS mutation. CONCLUSIONS: In TS, the loss of voltage-dependent inactivation is an upstream initiating event for arrhythmia phenotypes that are ultimately dependent on CaMKII activation.

An RNA Aptamer-Based Biomarker Platform Demonstrates High Soluble CD25 Occupancy by IL2 in the Serum of Follicular Lymphoma Patients.

Ligand-receptor complexes play a central role in mediating a range of processes in immunology and cancer biology. The ability to directly quantify the fraction of receptors occupied by a ligand in a given biospecimen, as opposed to assessing the concentration of ligand and receptor separately, could provide an additional and valuable clinical and research tool for assessing whether receptors are occupied by a ligand. To address this need, a biomarker platform was developed to quantify the fraction of receptors occupied by a ligand using pairs of RNA aptamers, where one aptamer binds preferentially to the unoccupied receptor and the other to the ligand-receptor complex. Bound aptamer was quantified using RT-qPCR colorimetric probes specific for each aptamer. The binding ratio of aptamer correlated with the fraction of receptors occupied by a ligand. This assay, termed as LIRECAP (LIgand-REceptor Complex-binding APtamer) assay, was used to determine the fraction of soluble CD25 occupied by IL2 in the serum from subjects with B-cell lymphoma. No correlation was found between the type of lymphoma and total soluble CD25 or IL2 independently. In contrast, the fraction of soluble CD25 occupied by IL2 was significantly higher in follicular lymphoma patient serum compared with diffuse large B-cell lymphoma patient serum. We conclude that this technology has the potential to serve as a high-throughput biomarker platform to quantify the fraction of receptors occupied by a ligand.

Delivery of Cell-Specific Aptamers to the Arterial Wall with an Occlusion Perfusion Catheter.

Current strategies to prevent restenosis following endovascular treatment include the local delivery of anti-proliferative agents to inhibit vascular smooth muscle cell (VSMC) proliferation and migration. These agents, not specific to VSMCs, are deposited on the luminal surface and therefore target endothelial cells and delay vascular healing. Cell-targeted therapies, (e.g., RNA aptamers), can potentially overcome these safety concerns by specifically binding to VSMC and inhibiting proliferation and migration. The purpose of this study was to therefore demonstrate the ability of a perfusion catheter to deliver cell-specific RNA aptamer inhibitors directly to the vessel wall. RNA aptamers specific to VSMCs were developed using an in vitro cell-based systematic evolution of ligand by exponential enrichment selection process. Two aptamers (Apt01 and Apt14) were evaluated ex vivo using harvested pig arteries in a pulsatile flow bioreactor. Local drug delivery of the aptamers into the medial wall was accomplished using a novel perfusion catheter. We demonstrated the feasibility to deliver aptamer-based drugs directly to the medial layer of an artery using a perfusion catheter. Such cell-specific targeted therapeutic drugs provide a potentially safer and more effective treatment option for patients with vascular disease.

RNA inhibitors of nuclear proteins responsible for multiple organ dysfunction syndrome.

The development of multiple organ dysfunction syndrome (MODS) following infection or tissue injury is associated with increased patient morbidity and mortality. Extensive cellular injury results in the release of nuclear proteins, of which histones are the most abundant, into the circulation. Circulating histones are implicated as essential mediators of MODS. Available anti-histone therapies have failed in clinical trials due to off-target effects such as bleeding and toxicity. Here, we describe a therapeutic strategy for MODS based on the neutralization of histones by chemically stabilized nucleic acid bio-drugs (aptamers). Systematic evolution of ligands by exponential enrichment technology identified aptamers that selectively bind those histones responsible for MODS and do not bind to serum proteins. We demonstrate the efficacy of histone-specific aptamers in human cells and in a murine model of MODS. These aptamers could have a significant therapeutic benefit in the treatment of multiple diverse clinical conditions associated with MODS.

Aptamers as Diagnostic Tools in Cancer.

Cancer is the second leading cause of death worldwide. Researchers have been working hard on investigating not only improved therapeutics but also on early detection methods, both critical to increasing treatment efficacy, and developing methods for disease prevention. The use of nucleic acids, or aptamers, has emerged as more specific and accurate cancer diagnostic and therapeutic tools. Aptamers are single-stranded DNA or RNA molecules that recognize specific targets based on unique three-dimensional conformations. Despite the fact aptamer development has been mainly restricted to laboratory settings, the unique attributes of these molecules suggest their high potential for clinical advances in cancer detection. Aptamers can be selected for a wide range of targets, and also linked with an extensive variety of diagnostic agents, via physical or chemical conjugation, to improve previously-established detection methods or to be used as novel biosensors for cancer diagnosis. Consequently, herein we review the principal considerations and recent updates in cancer detection and imaging through aptamer-based molecules.

Combination of Proteasome and Histone Deacetylase Inhibitors Overcomes the Impact of Gain-of-Function p53 Mutations.

Mutations in the "guardian of the genome" TP53 predominate in solid tumors. In addition to loss of tumor suppressor activity, a specific subset of missense mutations confers additional oncogenic properties. These "gain-of-function" (GOF) mutations portend poor prognosis across cancer types regardless of treatment. Our objective in this study was to identify novel therapeutic opportunities to overcome the deleterious effects of GOF TP53 mutants. Using gynecologic cancer cell lines with known TP53 mutational status, we established that treatment with a proteasome inhibitor induced cell death in cells with two recurrent GOF TP53 mutations (R175H and R248Q), and addition of a histone deacetylase inhibitor (HDACi) enhanced this effect. By contrast, p53-null cancer cells were relatively resistant to the combination. Proteasome inhibition promoted apoptosis of cells with TP53 GOF mutations, potentially through induction of the unfolded protein response. In line with the reported hyperstabilization of GOF p53 protein, cells treated with HDACi exhibited reduced levels of p53 protein. Together, these data form the basis for future clinical studies examining therapeutic efficacy in a preselected patient population with GOF TP53 mutations.

E-C coupling structural protein junctophilin-2 encodes a stress-adaptive transcription regulator.

Junctophilin-2 (JP2) is a structural protein required for normal excitation-contraction (E-C) coupling. After cardiac stress, JP2 is cleaved by the calcium ion-dependent protease calpain, which disrupts the E-C coupling ultrastructural machinery and drives heart failure progression. We found that stress-induced proteolysis of JP2 liberates an N-terminal fragment (JP2NT) that translocates to the nucleus, binds to genomic DNA, and controls expression of a spectrum of genes in cardiomyocytes. Transgenic overexpression of JP2NT in mice modifies the transcriptional profile, resulting in attenuated pathological remodeling in response to cardiac stress. Conversely, loss of nuclear JP2NT function accelerates stress-induced development of hypertrophy and heart failure in mutant mice. These data reveal a self-protective mechanism in failing cardiomyocytes that transduce mechanical information (E-C uncoupling) into salutary transcriptional reprogramming in the stressed heart.

Galaxy Workflows for Web-based Bioinformatics Analysis of Aptamer High-throughput Sequencing Data.

Development of RNA and DNA aptamers for diagnostic and therapeutic applications is a rapidly growing field. Aptamers are identified through iterative rounds of selection in a process termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). High-throughput sequencing (HTS) revolutionized the modern SELEX process by identifying millions of aptamer sequences across multiple rounds of aptamer selection. However, these vast aptamer HTS datasets necessitated bioinformatics techniques. Herein, we describe a semiautomated approach to analyze aptamer HTS datasets using the Galaxy Project, a web-based open source collection of bioinformatics tools that were originally developed to analyze genome, exome, and transcriptome HTS data. Using a series of Workflows created in the Galaxy webserver, we demonstrate efficient processing of aptamer HTS data and compilation of a database of unique aptamer sequences. Additional Workflows were created to characterize the abundance and persistence of aptamer sequences within a selection and to filter sequences based on these parameters. A key advantage of this approach is that the online nature of the Galaxy webserver and its graphical interface allow for the analysis of HTS data without the need to compile code or install multiple programs.

Cell-internalization SELEX: method for identifying cell-internalizing RNA aptamers for delivering siRNAs to target cells.

After a decade of work to address cellular uptake, the principal obstacle to RNAi-based therapeutics, there is now well-deserved, renewed optimism about RNAi-based drugs. Phase I and II studies have shown safe, strong, and durable-gene knockdown (80-90%, lasting for a month after a single injection) and/or clinical benefit in treating several liver pathologies. Although promising, these studies have also highlighted the need for robust delivery techniques to develop RNAi therapeutics for treating other organ systems and diseases. Conjugation of siRNAs to cell-specific, synthetic RNA ligands (aptamers) is being proposed as a viable solution to this problem. While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types. We describe a cell-based selection process for the rapid identification and characterization of RNA aptamers suited for delivering siRNA drugs into the cytoplasm of target cells. This process, termed "cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment)," entails the combination of multiple sophisticated technologies, including cell culture-based SELEX procedures, next-generation sequencing (NGS), and novel bioinformatics tools.

Identification of RNA aptamers that internalize into HPV-16 E6/E7 transformed tonsillar epithelial cells.

Human papillomavirus type 16 (HPV-16) associated oropharyngeal cancers are on a significant increase and better therapeutic strategies are needed. The HPV-16 oncogenes E6 and E7 are expressed in HPV-associated cancers and are able to transform human tonsillar epithelial cells (HTECs). We used cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select for RNA aptamers that entered into HPV-16 E6/E7-HTECs. After 12 rounds of cell-SELEX, a pool of aptamers was obtained that had significantly greater internalization capacity (~5-fold) into E6/E7-HTECs as compared to primary HTECs or fibroblasts. Analysis of individual aptamers from the pool indicated variable internalization into E6/E7-HTECs (1-8-fold as compared to a negative control). Most of the individual aptamers internalized into E6/E7 and primary HTECs with similar efficiency, while one aptamer exhibited ~3-fold better internalization into E6/E7-HTECs. Aptamers that internalize into cells may be useful for delivering therapeutic agents to HPV-16 associated malignancies.