Tailoring Modular Spherical Nucleic Acids for DNA and RNA Delivery

Sinegra, Andrew

doi:https://doi.org/10.21985/n2-x37v-v420

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Tailoring Modular Spherical Nucleic Acids for DNA and RNA Delivery

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Nucleic acids such as DNA or RNA of various lengths and structures have a wide scope of functions as therapeutic entities compared to conventional drugs. For instance, native and modified forms of nucleic acids can be used for gene silencing, genome editing, gene replacement, immune system modulation, and theranostics. While significant progress has been made, the extensive implementation of nucleic acid-based therapeutics has been limited by their ability to be effectively delivered at clinically relevant doses; to do so, their clearance from circulation must be prevented and their ability to cross biological barriers and enter target cells enhanced. Extensive modifications to both nucleic acid sequence and structure are being made to address this issue.Spherical nucleic acid (SNA) nanostructures, where oligonucleotides, DNA or RNA, are radially oriented in a densely packed fashion around a spherical nanoparticle template, have been utilized to overcome challenges in nucleic acid delivery. This three-dimensional arrangement of nucleic acids allows for efficacious nucleic acid delivery owing to its increased circulation time and resistance to degradation. These properties allow SNAs to accumulate in many cell types (measured in over 50 to date). The first SNAs were formed from inorganic nanoparticles, specifically gold nanoparticles (AuNPs), and since then, the possible design space of modifications to the nucleic acid sequences and the associated nanoparticle have been intensely explored. Indeed, many of the SNA properties mentioned above that have led to their utility in cellular and biological systems are maintained across many different nanoparticle types (i.e., regardless of the type of SNA core used). These include, but are not limited to: AuNPs, polymeric nanoparticles, liposomes, and proteins. The modular synthesis of SNAs and the diversity of the types of structures that can be engineered have opened new avenues to exploring structure-function relationships, especially in the case of organic nanoparticles, such as those made with lipid components (liposomes and lipid nanoparticles). This dissertation presents an investigation of how liposome and lipid nanoparticle (LNP)-based SNA structures can be tuned for the enhanced delivery and function of nucleic acids in vivo. In Chapter 1, the basis for this work is provided and previous types of SNAs, their applications, and their structures are analyzed. In Chapter 2, an investigation of how liposomal SNAs can be tuned for enhanced DNA delivery to major organs outside of the liver using hydrophobic anchors with different affinities for the liposomal nanoparticle core is described. In Chapter 3, a strategy to reprogram the function of existing LNP formulations by targeting them with DNA, forming LNP-SNAs, is offered. LNP-SNA formulations were optimized in cellular assays to attain the greatest cytosolic delivery, and their function was assessed in wild-type mice in the context of mRNA delivery. In Chapter 4, the relationships between LNP-SNA structure and nanoparticle distribution and function in vivo are assessed. In Chapter 5, future directions for characterizing lipid-based SNAs as well as expanding their applications are discussed. The overall objective of these studies was to build a foundation for designing and synthesizing nanoparticle structures for the delivery of functional DNA or RNA inside cells.

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