493. Aptamer-Mediated Targeted Delivery of Large RNA
Authors: Khalid K. Alam, David Porciani, Kyle J. Hill, Donald H. Burke
Journal: Molecular Therapy (2015)

Cell penetrating RNAs bind to cell surface receptors and internalize through endocytotic pathways. These delivery molecules are increasingly investigated for cell type targeted delivery of a wide variety of payloads, such as small molecule drugs, imaging agents and small interfering RNAs. Although larger oligonucleotides also have therapeutic and regulatory potential, their aptamer-mediated delivery into cells has not yet been reported. Recent advances in the selection and design of fluorescent aptamers allow for the tracking and visualization of RNA intracellularly and raise the possibility of directly monitoring delivery of large oligonucleotide payloads. Here we report on a plug-and-play platform for the targeted delivery of large functional RNA. Various cell-type specific targeting oligos, such as the anti-transferrin receptor aptamer, were annealed to variants of the fluorescent RNAs, Spinach and Broccoli, either through direct base-pairing of extension sequences or through a bridging oligo designed to release the payload in reducing environments. The size of the RNA payload (30-164 kDa) was explored through multimerization of the aptamers and was accomplished through several designs. Low signal intensity associated with the Spinach2 aptamer was partially overcome by dimerization of a minimal form of the aptamer, which yielded a nearly 2-fold increase in fluorescence per RNA transcript. The relatively small size of the dimeric Broccoli aptamer facilitated engineering of a tetrameric aptamer by inserting dimers into each end of a thermodynamically stable three-way junction RNA, and a much larger octomer by incorporating two tetramers on a single transcript. In all cases, multimerization of aptamer payloads resulted in significantly enhanced fluorescence. Formation of the self-assembling aptamer-aptamer complexes was confirmed through electrophoretic mobility shift assays and revealed that the fraction of fully annealed product varied considerably among designs and may account for some variations observed in previous studies for siRNA efficacy. Complex functionality was validated with spectrofluorometry, which reinforced the importance of designing RNA components to avoid misfolding that can abrogate functionality. Fluorescence confocal microscopy was used to detect delivery of functional RNA to a range of human and mice cells and demonstrated specificity of targeting aptamers for their intended cell-types. Delivered complexes exhibited punctate intracellular distribution, suggesting their localization in the endosomes. Our findings demonstrate that large functional RNAs can be delivered to living cells and may present the opportunity to deliver other large oligos, such as long non-coding RNAs and CRISPR/Cas9 guide RNA, to targeted cell types. Importantly, because fluorescent signal is only evident while the RNA remains intact and correctly folded, this strategy provides a powerful method to monitor the real-time integrity, delivery and localization of large and functional RNA cargoes.