Characterization of Cellular Internalization Pathways for CPP-Mediated Oligonucleotide Delivery
The methods for evaluating internalization pathways of cellular CPP-mediated ON delivery utilizing a pre-mRNA splice correction assay and fluorescence-based quantification are described. Examples for characterization of CPP uptake routes, employing various endocytosis inhibitors, and special treatment conditions are demonstrated. The methods are developed to characterize cellular delivery of pre-mRNA splice switching peptide nucleic acids conjugated to CPPs by disulfide bond.
Key wordsPre-mRNA splicing Oligonucleotide delivery Internalization pathways Endocytosis inhibitors
pLuc705-HeLa cells were kindly provided by R. Kole and B. Lebleu. This work was supported by the Swedish Science Foundation (VR-NT and VR-MED), the Knut and Alice Wallenberg Foundation, the Swedish Governmental Agency for Innovation Systems (VINNOVA-SAMBIO 2006), and the Swedish Center for Biomembrane Research.
- 15.Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hökfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K., Ossipov, M., Koshkin, A., Jakobsen, N., Skouv, J., Oerum, H., Jacobsen, M. H., and Wengel, J. (2000) Potent and Nontoxic Antisense ONs Containing Locked Nucleic Acids. Proc. Natl. Acad. Sci. U. S. A. 97, 5633–5638.CrossRefPubMedGoogle Scholar