Cell-Penetrating Aptamers Targeting Sub-Cellular Compartments

靶向亚细胞区室的细胞穿透适体

• 批准号: 9764414
• 负责人: LOUIS JAMES MAHER
• 金额: $ 29.44万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764414
• 关键词: Address; Animals; Base Sequence; Binding; CD69 antigen; Cations; Cell Nucleus; Cell surface; Cells; Code; Complex; DNA; DNA Ligases; Development; Endocytic Vesicle; Exposure to; Fishes; Formulation; Future; Goals; Home environment; Homing; In Vitro; Libraries; Ligase; Lipids; Medicine; Membrane; Membrane Fusion; Methods; Modernization; Modification; Mus; Nuclear; Nucleic Acids; Organelles; Pathway interactions; Penetration; Property; Reagent; Recipe; Rewards; System; Testing; Tissues; Vesicle; aptamer; base; design; enzyme activity; improved; in vivo; nanomedicine; novel strategies; nucleic acid delivery; sensor; subcellular targeting; uptake

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.

靶向亚细胞区室的细胞穿透适体 摘要：现代纳米医学的一个未满足的需求是有效递送核酸的方法 以及相关的货物进入活组织中的相关亚细胞区室。当细胞暴露于 核酸或核酸/阳离子脂质复合物，常规方法导致摄取到膜中， 结合的囊泡（仍然拓扑地在细胞外）或不加选择的膜融合。而且很多 载体脂质制剂是有毒的并且在体内的使用有限。虽然体外选择的力量（SELEX） 先前被应用于选择结合细胞或获得优先囊泡摄取的核酸序列 进入特定的细胞或组织，尚未设想具有细胞内靶向的囊泡逃逸。我们有 开发了一种新的方法来实现这一目标。我们应用体外选择来鉴定核酸适体， 有效地进入特定的亚细胞区室通过选择的序列， 依赖于特定细胞内酶活性的修饰。我们发现这种“奖励”方法 可以鉴定出具有显著改善的细胞核递送的裸DNA适体（“亲核的 适体）。该方法将被扩展到识别不同组织的细胞穿透适体， 活老鼠能够囊泡逃逸和亚细胞区室递送的奖励适体序列 开辟了一个新的机会领域。在未来，也许有可能扩展这种奖励方法，以确定 靶向其他亚细胞区室的序列。提出了四个具体目标来检验假设 靶向特定亚细胞区室的细胞穿透DNA适体可以通过以下方式艾德： 选择由细胞器特异性酶活性修饰的艾德分子，并且这种归巢适体 可以有效地将货物运送到细胞和组织。目的1将继续我们的选择嗜核（核- 归巢）DNA适体，为靶向其他亚细胞区室奠定基础。目标2将寻求 了解嗜核DNA适体的核传递机制。目标3将探索 亲核DNA适体以引导货物递送到细胞中。目标4将在体内扩展这种奖励方法， 在小鼠中开发组织特异性嗜核DNA适体文库。