Improving Biological Properties of Spherical Nucleic Acids Through Structural Control

Vasher, Matthew Kuo

doi:https://doi.org/10.21985/n2-w07j-en07

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Improving Biological Properties of Spherical Nucleic Acids Through Structural Control

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Nucleic acid therapeutics can be drug molecules with high programmability, minimal off-target effects, and the capability to address “undruggable” targets for diseases. In addition, each time a new drug is needed, one needs to only change the sequence as opposed to finding an entirely new structure. One nucleic acid type in particular, small interfering RNA (siRNA), has shown particular promise as a therapeutic. siRNAs are short, double-stranded RNA molecules that can be designed to silence any gene of interest. Due to their programmability, specificity, and potency, siRNAs have tremendous potential as therapeutics for cancers, autoimmune disorders, and any other diseases that are driven by the overexpression of a gene. However, despite over two decades of research, siRNAs have yet to reach widespread clinical use, with the first siRNA drug receiving FDA approval in 2018 and only four more being approved since then, all for treating the liver. The clinical success of siRNAs has been severely restricted by poor stability and delivery: unmodified siRNAs are rapidly degraded in biological fluids by nucleases, have poor pharmacokinetics, and cannot enter cells without transfection agents.To overcome these limitations, siRNAs can be radially arranged around a nanoparticle core to form a spherical nucleic acid (SNA). siRNA-based SNAs (siRNA-SNAs) gain unique properties that linear siRNAs lack, such as resistance to nuclease degradation and efficient entry into cells. At the start of my PhD, all published siRNA-SNAs followed a prototypical design: thiolated passenger strands were attached to a ~13-nm gold nanoparticle core, and complementary guide strands were hybridized to the passenger strands. Prototypical siRNA-SNAs have shown some success in mouse models of disease and even progressed to a first-in-human clinical trial, but they are limited in their performance and widespread applicability. Important biological properties critical to the efficacy of the siRNA-SNA include structural stability, nuclease resistance, biocompatibility, pharmacokinetics, cellular uptake, cytosolic delivery, and therapeutic activity, and all of these properties are limited by the prototypical siRNA-SNA’s structure. This dissertation investigates how structural changes to the siRNA-SNA can be used to improve its biological properties. Chapter 1 introduces siRNA, current delivery strategies, prototypical siRNA-SNAs, and the structure-function relationships of SNAs. In Chapter 2, a hairpin-like architecture for attaching siRNAs to the nanoparticle core is introduced, leading to improved structural stability, nuclease resistance, biocompatibility, cellular internalization, and therapeutic activity. In Chapter 3, the effect of core size on SNA behavior is investigated, with the finding that an ultrasmall 1.4-nm gold nanocluster core improves the SNA’s drug-to-carrier ratio, pharmacokinetics, and cellular uptake. In Chapter 4, lipid-based cores are explored as a biocompatible alternative to gold-based cores for siRNA-SNAs, leading to the design of next-generation siRNA-SNAs with improved biocompatibility, potential suitability as an ocular drug, and efficient cytosolic delivery. Finally, Chapter 5 concludes the dissertation with a summary of the findings and a description of future work in the development of next-generation SNAs. While this work primarily focuses on siRNA-SNAs, many of the structure-function relationships characterized herein will apply to SNAs composed of other nucleic acids as well. Overall, this dissertation shows that control over chemical structure can be used to significantly enhance the biological properties of SNAs, improving their therapeutic suitability. These findings will play an important role in the design of next-generation SNAs as well as drive the progression of siRNAs toward widespread clinical use.

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