Cell-penetrating aptamers targeting sub-cellular compartments ABSTRACT: An unmet need in modern nanomedicine is a method for ef?cient 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 speci?c 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 ef?ciently enter speci?c sub-cellular compartments by selecting for sequences that undergo enzymatic modi?cation dependent on speci?c 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 speci?c for different tissues in living mice. Rewarding aptamer sequences capable of vesicle escape and sub-cellular compartment delivery opens a new ?eld of opportunities. In the future it may be possible to extend this reward approach to identify sequences that target other sub-cellular compartments. Four speci?c aims are proposed to test the hypotheses that cell-penetrating DNA aptamers targeting speci?c sub-cellular compartments can be identi?ed by selecting molecules modi?ed by organelle-speci?c enzyme activities, and that such homing aptamers can ef?ciently 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-speci?c karyophilic DNA aptamers in mice.
Cell-penetrating aptamers targeting sub-cellular compartments Medicines of the future will contain code recipes and other complex molecules that change the ways that cells grow and divide. These medicines are very different from current medicines because they are large and complicated, and created from materials that do not naturally cross into the cells of the body. This proposal describes an exciting new method based on the idea that we don't know how to design molecules that can sneak into cells, but if we know how to make trillions of different molecules at random and reward the very few that happen to achieve the goal, we can then ?sh them out, copy them, and exploit their special properties.
