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 reagen...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2008-10, Vol.105 (41), p.15908-15913
Main Authors: Chen, Chi-hong B, Dellamaggiore, Kenneth R, Ouellette, Christopher P, Sedano, Cecilia D, Lizadjohry, Meikana, Chernis, George A, Gonzales, Michelle, Baltasar, Francis E, Fan, Audrey L, Myerowitz, Rachel, Neufeld, Elizabeth F
Format: Article
Language:English
Subjects:
Animals
Aptamers, Nucleotide - pharmacology
Biological Sciences
Cell lines
Cells
Cells, Cultured
Endocytosis
Endothelial cells
Enzymes
Enzymes - metabolism
Fibroblasts
Fibroblasts - cytology
Fibroblasts - metabolism
Humans
Iduronidase - metabolism
Lysosomes
Lysosomes - enzymology
Mice
Nucleotide aptamers
Proteins
Receptors
Receptors, Transferrin - metabolism
Rodents
Species Specificity
Streptavidin
Transferrin receptors
Transferrins
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Summary: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, α-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-α-L-iduronidase-aptamer conjugate was taken up in saturable manner by α-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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0808360105