Development of DNA origami-based tools for cancer treatment

Abstract: DNA has been used as material for the assembly of objects at different scales. Particularly, the introduction of DNA origami has been inspiring the design and construction of many different versions of DNA nanostructures for, especially, biomedical applications. DNA origami nanostructures are showing unique advantages, including structural homogeneity, addressability, biocompatibility, and capacity to carry pharmaceuticals or biomolecules, for the development of future cancer therapeutics. To fully unlock their potential, however, they need to be tailored based on physiological and pathological molecular environments which they interact with. In this thesis, we develop a few functionalized DNA origami nanostructures to investigate specific questions of cancer biology or to overcome challenges of cancer immunotherapy. In PAPER I, we compare the physical characteristics of a compact lattice-based rod and a wireframe-styled rod and revealed how they interact with cell spheroid tissue models (CSTMs). Our data indicate that the wireframe structure, which has a lower local material density in design, has higher local deformability than the lattice-based structure. We reveal that these physical differences play important roles in the interaction between DNA origami nanostructures and human cancer cells, showing that wireframe rods are more likely to stay on the cell membrane, rather than being internalized, and this facilitates their deeper penetration into CSTMs. These observations tell us that DNA origami design methods should be carefully considered in DNA origami-based drug delivery applications. In PAPER II, to explore the nanoscale clustering effect of death receptor 5 (DR5) on human breast cancer cells, we develop flat sheet-like DNA origami nanostructures and functionalize them with the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- mimicking peptides which can recognize and bind with DR5, in differently sized hexagonal patterns. Experiments of cancer cells and DNA origami incubation show that apoptosis can be precisely controlled when we vary the size of peptide patterns between the range of 6 nm to 26 nm. Interestingly, our data indicate that the interpeptide distance for effective apoptosis is sub-10 nm. Our findings highlight the potency of precise spatial patterning of ligands on apoptosis signaling. In PAPER III, to limit cytotoxicity of the sub-10 nm peptide pattern, which we have screened out in the work of PAPER II, only to tumors, we design a three-dimensional DNA origami nanostructure containing a 6 nm wide and 12 nm deep cavity and use it to hide but display the peptide pattern according to the acidity of the tissue microenvironment. Peptide display is achieved by the protonation-triggered formation of the DNA triplex, during which a singlestranded DNA extension from the complementary strand of a mini-scaffold DNA wraps back to the mini-scaffold duplex. By varying the AT percentage in the mini-scaffold DNA, we can control the triggering pH for the formation of the DNA triplex. We demonstrate the safety of the DNA origami under physiological pH (pH 7.4) for non-cancer cells and its cytotoxicity to cancer cells under the pH of solid tumors (pH 6.5). In PAPER IV, to mimic functions of T cell engagers but mitigate corresponding adverse effects mainly caused by “on-target, off-tumor” immune activation and cytokine release syndrome, we develop a wireframe DNA origami based-nanorobot: a double-layered barrellike origami with antibodies inside under its closed status. When the DNA nanorobot presents as its open configuration, internal antibodies get exposed, functioning to engage T cells with cancer cells and activate T cell immune killing. By spatiotemporally controlling the opening of the DNA nanorobot via signals from cancer cells, tumor microenvironment, or external stimulus, we aim to selectively redirect the cytotoxicity of T cells to solid cancers and substantially mitigate corresponding adverse effects of current T cell engagers-based cancer immunotherapy.

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