Aptamer-based endocytosis of a lysosomal enzyme

Abstract

Enzyme replacement therapy for lysosomal storage diseases is currently based on endocytosis of lysosomal enzymes via the mannose or mannose 6-phosphate receptors. We are developing a technology for endocytosis of lysosomal enzymes that depends on generic, chemically conjugated reagents. These reagents are aptamers (singlestranded nucleic acid molecules) selected to bind to the extracellular domain of the mouse transferrin receptor. After selection, an RNA aptamer and a DNA aptamer were modified with biotin and linked to dye-labeled streptavidin for detection by confocal microscopy. Aptamer-streptavidin conjugates showed saturable uptake into mouse fibroblasts (Ltk ؊ cells), which could be inhibited by an excess of free aptamer but not by tRNA, calf thymus DNA, or transferrin. The RNA aptamer-streptavidin conjugate was mouse-specific, as human cells (293T) did not take it up unless first transfected with the mouse transferrin receptor. Some streptavidin separated from the recycling pathway of transferrin and colocalized with lysosomes. After characterization in the model system, the DNA aptamer was conjugated to a lysosomal enzyme, ␣-L-iduronidase, from which mannose 6-phosphate had been removed. The aptamer had been modified by attachment of terminal glycerol for oxidation by periodate and reaction of the resulting aldehyde with amino groups on the protein. Dephospho-␣-L-iduronidase-aptamer conjugate was taken up in saturable manner by ␣-L-iduronidase-deficient mouse fibroblasts, with half-maximal uptake estimated as 1.6 nM. Endocytosed enzyme-aptamer conjugate corrected glycosaminoglycan accumulation, indicating that it reached lysosomes and was functional in those organelles. Both uptake and correction were inhibited by unconjugated aptamer, confirming the role of the aptamer in receptor-mediated endocytosis.lysosomal storage disease ͉ transferrin receptor

Aptamers as Promising Molecular Recognition Elements for Diagnostics and Therapeutics in the Central Nervous System

“…Transferrin receptors (TfRs) are particularly interesting aptamer targets for delivery strategies as TfRs are highly expressed on endothelial cells and are known to be involved in transcytosis across the BBB. Both RNA (FB4) and DNA (GS24) aptamers that bind to mouse TfRs have been selected [143]. Each of the FB4 and GS24 aptamers has shown cellular internalization.…”

Aptamer photoregulation in vivo

“…Aptamers can be covalently bound to a wide variety of therapeutic agents, including chemotherapeutics or enzymes (45,46), siRNA (47,48), or drug-loaded nanocarriers (36,49), to provide targeted drug delivery. The spatiotemporal control of drug delivery afforded by our approach may enhance the efficacy and therapeutic index of many drugs.…”

DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro

“…We found that the FITC fluorescence of aptamers 41, 43, and 32 appeared to be localized within the nuclei, whereas aptamers 41 and 43 were also present in the cytoplasm. The transportation of the aptamers into the cells may have occurred through endocytosis [25,26] ; therefore, we hypothesized that some of these aptamers may recognize EGFRvIII. Aptamer 47 also localized to the cell surface.…”