Aptamer-based endocytosis of a lysosomal enzyme.

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 (single-stranded 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, alpha-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-alpha-L-iduronidase-aptamer conjugate was taken up in saturable manner by alpha-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.

Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3.

Human epidermal growth factor receptor-3 (HER3) is a member of the type I receptor tyrosine kinase family. Several members of this family are overexpressed in various carcinomas. Specifically, HER2 is found to be overexpressed in 20-30% of breast cancers. In contrast to epidermal growth factor receptor or HER2, the kinasedeficient HER3 self-associates readily at low nanomolar concentrations and in the absence of its ligands, various isoforms of heregulin (hrg). Binding of hrg disrupts HER3 oligomerization and leads to the formation of signaling-competent heterodimers, preferentially with HER2. Elevated levels of HER3 contribute to increased drug resistance observed in HER2-overexpressing cells. We have used the SELEX (systematic evolution of ligands by exponential enrichment) methodology to select RNA aptamers against the oligomeric state of the extracellular domains of HER3 (HER3ECD, monomeric molecular mass 82,000 Da). One of the aptamers, A30, binds with high affinity to a limited number of binding sites in the oligomeric state of HER3ECD. Binding of A30 and hrg are not competitive. Instead, the disruption of HER3 oligomers by hrg results in an approximately 10-fold increase in total binding sites, but the newly created binding sites are of lower affinity. High-affinity binding of A30 inhibits hrg-dependent tyrosine phosphorylation of HER2 and the hrg-induced growth response of MCF7 cells. As an example of an aptamer against a large macromolecular protein complex, A30 can serve as a tool for the analysis of receptor interactions and may serve as a lead compound for the development of inhibitors against overexpressed receptor tyrosine kinases in carcinomas.