Wilbanks, Brandon

Abstract Despite recent advances in the understanding of brain tumor pathophysiology, challenges associated with tumor location and characteristics have prevented significant improvement in neuro-oncology therapies. Aptamers are short, single-stranded DNA or RNA oligonucleotides that fold into sequence-specific, 3-dimensional shapes that, like protein antibodies, interact with targeted ligands with high affinity and specificity. Aptamer technology has recently been applied to neuro-oncology as a potential approach to innovative therapy. Preclinical research has demonstrated the ability of aptamers to overcome some obstacles that have traditionally rendered neuro-oncology therapies ineffective. Potential aptamer advantages include their small size, ability in some cases to penetrate the blood-brain barrier, inherent lack of immunogenicity, and applicability for discovering novel biomarkers. Herein, we review recent reports of aptamer applications in neuro-oncology including aptamers found by cell- and in vivo- Systematic Evolution of Ligands by Exponential Enrichment approaches, aptamer-targeted therapeutic delivery modalities, and aptamers in diagnostics and imaging. We further identify crucial future directions for the field that will be important to advance aptamer-based drugs or tools to clinical application in neuro-oncology.

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.

We previously reported the in vitro selection and characterization of a DNA aptamer capable of stimulating remyelination in a mouse model of multiple sclerosis. This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro . Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5′ half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5′ half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro , providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.