Cell-Penetrating Aptamers Targeting Sub-Cellular Compartments

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Cell-penetrating aptamers targeting sub-cellular compartments ABSTRACT: An unmet need in modern nanomedicine is a method for efficient delivery of nucleic acids and related cargo into relevant sub-cellular compartments in living tissues. Upon exposure of cells to nucleic acids or nucleic acid/cationic lipid complexes, conventional methods result in uptake into membrane- bound vesicles (still topologically outside of the cell) or indiscriminate membrane fusion. Moreover, many carrier lipid formulations are toxic and of limited use in vivo. While the power of in vitro selection (SELEX) has been previously applied to select nucleic acid sequences that bind cells or gain preferential vesicular uptake into specific cells or tissues, vesicular escape with intracellular targeting has not been envisioned. We have developed a novel approach to this goal. We apply in vitro selection to identify nucleic acid aptamers that efficiently enter specific sub-cellular compartments by selecting for sequences that undergo enzymatic modification dependent on specific intracellular enzyme activities. We show that this "reward" approach can identify naked DNA aptamers with substantially improved delivery to the cell nucleus (“karyophilic” aptamers). The method will be extended to identify cell-penetrating aptamers specific for different tissues in living mice. Rewarding aptamer sequences capable of vesicle escape and sub-cellular compartment delivery opens a new field of opportunities. In the future it may be possible to extend this reward approach to identify sequences that target other sub-cellular compartments. Four specific aims are proposed to test the hypotheses that cell-penetrating DNA aptamers targeting specific sub-cellular compartments can be identified by selecting molecules modified by organelle-specific enzyme activities, and that such homing aptamers can efficiently deliver cargo to cells and tissues. Aim 1 will continue our selection of karyophilic (nucleus- homing) DNA aptamers, setting the stage for the targeting of other sub-cellular compartments. Aim 2 will seek to understand the mechanism of nuclear delivery of karyophilic DNA aptamers. Aim 3 will explore the ability of karyophilic DNA aptamers to direct cargo delivery into cells. Aim 4 will extend this reward approach in vivo to develop a library of tissue-specific karyophilic DNA aptamers in mice.