Cell-Internalization SELEX: Method for Identifying Cell-Internalizing RNA Aptamers for Delivering siRNAs to Target Cells
After a decade of work to address cellular uptake, the principal obstacle to RNAi-based therapeutics, there is now well-deserved, renewed optimism about RNAi-based drugs. Phase I and II studies have shown safe, strong, and durable-gene knockdown (80–90 %, lasting for a month after a single injection) and/or clinical benefit in treating several liver pathologies. Although promising, these studies have also highlighted the need for robust delivery techniques to develop RNAi therapeutics for treating other organ systems and diseases. Conjugation of siRNAs to cell-specific, synthetic RNA ligands (aptamers) is being proposed as a viable solution to this problem. While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types. We describe a cell-based selection process for the rapid identification and characterization of RNA aptamers suited for delivering siRNA drugs into the cytoplasm of target cells. This process, termed “cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment),” entails the combination of multiple sophisticated technologies, including cell culture-based SELEX procedures, next-generation sequencing (NGS), and novel bioinformatics tools.
Key wordsRNA aptamers Systematic evolution of ligands by exponential enrichment (SELEX) Cell-internalization SELEX Cell-targeted aptamers Next-generation sequencing (NGS) Bioinformatics Quantitative PCR
- 14.Dassie JP, Liu XY, Thomas GS, Whitaker RM, Thiel KW, Stockdale KR, Meyerholz DK, McCaffrey AP, McNamara JO II, Giangrande PH (2009) Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors. Nat Biotechnol 27:839–849PubMedCrossRefPubMedCentralGoogle Scholar
- 15.Wheeler LA, Vrbanac V, Trifonova R, Brehm MA, Gilboa-Geffen A, Tanno S, Greiner DL, Luster AD, Tager AM, Lieberman J (2013) Durable knockdown and protection from hiv transmission in humanized mice treated with gel-formulated cd4 aptamer-siRNA chimeras. Mol Ther 21:1378–1389PubMedCrossRefPubMedCentralGoogle Scholar