Wilbanks, Brandon A

Abstract Glioblastoma (GBM) is an aggressive, high-grade glioma with a near-universally fatal prognosis. Therapeutic failure is often attributed to the highly selective blood brain barrier (BBB), the diffuse infiltrative nature of the tumor, and the marked intratumoral heterogeneity of GBM. Although antibody drug conjugates ADCs have shown promise for high grade gliomas such as GBM, efficacy is limited by ADC size. Aptamers—short, synthetic, single-stranded DNA or RNA molecules—can be ∼6-fold lower in molecular weight than IgG antibodies and have the potential to cross the intact BBB. Unlike other nucleic acid-based therapies, aptamer function arises from three-dimensional shape rather than genetic coding. Here we aim to replace the targeting component of the ADC paradigm with a DNA aptamer, thus creating an aptamer-drug conjugate (ApDC). We employed in vivo SELEX using an orthotopic patient-derived xenograft (PDX) GBM mouse model and a vast (∼100 trillion 80-mer sequences) ApDC library. We report the results from this first in vivo ApDC selection of its kind. We characterize target tissue binding ex vivo , cell association, biodistribution, and pharmacokinetics from this selection. This study exemplifies an unbiased approach to a problem that rational design has yet to overcome, offering a new direction for GBM therapeutic development.

ABSTRACT Cellular senescence is an irreversible form of cell‐cycle arrest caused by excessive stress or damage. While various biomarkers of cellular senescence have been proposed, there are currently no universal, stand‐alone indicators of this condition. The field largely relies on the combined detection of multiple biomarkers to differentiate senescent cells from non‐senescent cells. Here we introduce a new approach: unbiased cell culture selections to identify senescent cell‐specific folded DNA aptamers from vast libraries of trillions of random 80‐mer DNAs. Senescent mouse adult fibroblasts and their non‐senescent counterparts were employed for selection. We demonstrate aptamer specificity for senescent mouse cells in culture, identify a form of fibronectin as the molecular target of two selected aptamers, show increased aptamer staining in naturally aged mouse tissues, and demonstrate decreased aptamer staining when p16 expressing cells are removed in a transgenic INK‐ATTAC mouse model. This work demonstrates the value of unbiased cell‐based selections to identify new senescence‐specific DNA reagents.

Abstract Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor of adults. Current therapeutic options yield dismal prognoses that have remained essentially unchanged over nearly two decades. Diffuse growth patterns, high intratumoral heterogeneity, and variable blood–brain barrier integrity limit treatment efficacy, creating challenges that rational small molecule design has not overcome. Antibody–drug conjugates have shown some promise, leading us to hypothesize that smaller folded DNA aptamers, developed in vivo via principles of natural selection, might eventually have advantages for drug delivery. Here, we document the first in vivo DNA aptamer selection involving an orthotopic patient-derived xenograft GBM mouse model to identify tumor-homing DNA aptamers. We demonstrate the preferential accumulation of these aptamers in the tumor relative to other tissues 4 h after intraperitoneal injection. The aptamers can be detected by quantitative polymerase chain reaction, fluorescent tumor staining, and stain GBM tumor section from untreated mice and the GBM tumor cells in culture. Two of three candidates are selective for the target cell line in vitro and do not bind other human tumor cells. In vivo selection of tumor-specific DNA aptamers demonstrates a novel approach for diagnostics or toxin delivery that might allow for the development of individualized therapies.

AbstractIn humans, loss of heterozygosity for defective alleles of any of the four subunits of mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH, also Complex II of the electron transport chain) can lead to paraganglioma tumors in neuroendocrine cells. With the goal of developing mouse models of this rare disorder, we have developed various SDH conditional loss strategies. Based on recent lineage tracing studies, we hypothesized that conditional SDHC loss in early embryogenesis during migration of primordial neural crest cells that form the susceptible chromaffin cells of the adrenal medulla might induce paraganglioma. We triggered low levels of detectable SDHC loss in Sox10+ cells at E11.5 of mouse development. We report that, rather than developing adrenal medulla paraganglioma (pheochromocytoma), offspring survived with evidence of neural crest cell dysfunction. Phenotypes included mild lower extremity gait anomalies suggestive of neural tube closure defects and patches of unpigmented fur consistent with neural crest‐derived melanocyte dysfunction. These defects were not observed in mice lacking Sdhc knockout. Our results add to existing data suggesting that, unlike humans, even early embryonic (Sox10‐driven) SDHx loss is inadequate to trigger paraganglioma in mice of the genetic backgrounds that have been investigated. Instead, low levels of tricarboxylic acid cycle‐deficient neural crest cells cause mild developmental defects in hind limb and melanocyte function. This new model may be of interest for studies of metabolism during early neural crest cell development.

Abstract The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody.

A fascinating class of familial paraganglioma (PGL) neuroendocrine tumors is driven by the loss of the tricarboxylic acid (TCA) cycle enzyme succinate dehydrogenase (SDH) resulting in succinate accumulation as an oncometabolite and other metabolic derangements. Here, we exploit a Saccharomyces cerevisiae yeast model of SDH loss where accumulating succinate, and possibly reactive oxygen species, poison a dioxygenase enzyme required for sulfur scavenging. Using this model, we performed a chemical suppression screen for compounds that relieve dioxygenase inhibition. After testing 1280 pharmaceutically active compounds, we identified meclofenoxate HCl and its hydrolysis product, dimethylaminoethanol (DMAE), as suppressors of dioxygenase intoxication in SDH-loss yeast cells. We show that DMAE acts to alter metabolism so as to normalize the succinate:2-ketoglutarate ratio, improving dioxygenase function. This study raises the possibility that oncometabolite effects might be therapeutically suppressed by drugs that rewire metabolism to reduce the flux of carbon into pathological metabolic pathways.

AbstractRecent advances in peroxidase‐mediated biotin tyramide (BT) signal amplification technology have resulted in high‐resolution and subcellular compartment‐specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H2O2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence‐independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.