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DNA origami suit up to thwart molecular villains

Compact and protected DNA nanostructures enable enhanced therapeutic delivery into cells

By Eriona Hysolli

(BOSTON) – New and improved drugs and larger ‘biologics,’ such as monoclonal antibodies, are constantly being developed and tested as treatments for a wide range of human diseases. However, the delivery of these therapeutics is challenging because they are often poorly soluble in water, cannot easily pass cellular and organ barriers, have a short half-life, cause toxicity and immunogenicity, and are quickly degraded in the body’s extra- and intracellular milieus.

Fluorescent image of human bone-derived dendritic cells that have internalized large barrel-shaped DNA origami. Cellular nuclei are stained in blue and DNA origami in pink. Credit: Wyss Institute at Harvard University

To overcome these challenges, researchers have developed nanoparticles, or small molecular carriers, that are designed to deliver just the necessary amount of drug to the desired site with minimal toxicity and high efficiency. These nanoparticles can be made of protein, DNA, hydrogels, different types of polymers, metals, or lipids, and their surfaces can be chemically modified to improve their delivery. Frontrunners in the targeted delivery arena are DNA Nanoparticles (DNs), such as DNA origami, which are composed of a long single-strand of DNA or scaffold that folds into any one of a variety of shapes and sizes when it binds to hundreds of short DNA strands, called staples. The advantage of DNs is their ability to fold into any custom shape that can be designed using established computer programs, the ever decreasing cost of DNA synthesis, and the high degree of scalability in generating trillions of copies of these designs. But two essential questions have remained unanswered. How stable are the DNA origami inside living cells, and how efficiently can different cell types of the body take them up?

Now, a team of scientists at the Wyss Institute for Biologically Engineering at Harvard University has addressed both questions in two recent publications that bring the use of DNA origami as delivery vessels for drugs and therapeutic biomolecules closer to reality.

“What we know about DNA origami is that they quickly degrade in the low salt conditions found under physiological conditions in bodily fluids or the inside of cells, and in the presence of DNA nucleases, which are enzymes that are quick to take these structures apart”, says Maartje M.C. Bastings, Ph.D., a former postdoctoral fellow at the Wyss Institute and currently an Assistant Professor at École Polytechnique Fédérale de Lausanne who is one of the co-first authors of both publications appearing in Nature Communications and Nano Letters. High concentrations of positively charged divalent cations like magnesium are required to stabilize DNA origami due to the repelling electrostatic interactions between the negatively charged groups found in the backbone of DNA. However, this is not easily achievable in physiological fluids or inside cells where the concentration of the positively charged ions is smaller than what is required for origami stability. First, the Wyss scientists set out to probe the degradation of DNA origami in the presence of low salt buffers as well as DNA nucleases and found, as they expected, that their integrity was severely compromised. The team then designed a strategy that would allow them to stabilize and protect the DNA origami against these conditions. When they coated the DNA origami with a polymer of ten of the natural amino acid lysine (oligolysine) linked to polyethylene glycol (PEG), the stability of the origami in low salt and aggressive nuclease conditions improved significantly. The scientists went on to successfully test the shielded DNA origami inside living cells and mice, and showed in fluorescence experiments that these DNs were efficiently taken up  and remained intact inside them.

The next challenge that the Wyss team decided to tackle was to determine which shapes and sizes of the coated DNs would provide the most effective delivery into a variety of cell types. Shape, size and compactness of the origami are key factors for efficient cellular uptake. “A great analogy on how origami work is the magic snake toy that can be manipulated into different conformations”, says William Shih, Ph.D., Founding Core Faculty at the Wyss Institute, and Professor in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School and the Department of Cancer Biology at the Dana-Farber Cancer Institute. “We took advantage of the exquisite self-assembly, control of spacing, and reproducibility of DNA origami over other nanoparticles and folded DNA into nine main shape types and sizes. We found that, for particles of the same overall mass, the ones that are more compact are preferably internalized,” continued Shih. The team tested hollow and solid DNA origami rods, barrels, rings and blocks to test uptake kinetics in human endothelial, dendritic and embryonic kidney cells. All three cell types preferentially took up DNA origami with block and barrel shapes, and interestingly the dendritic cells, which are normally the vacuum cleaners of our bodies, had a great capacity to continuously internalize DNs compared to other cell types. This study shed important light in engineering the right shape of DNA origami for the desired type of target cell.

Computer models of DNA origami shapes built and tested by the Wyss scientists. Large shapes colored blue and small shapes colored orange. Credit: Wyss Institute at Harvard University

The Wyss scientists hope to take advantage of the fine tuning of origami spacing to crosslink ligands that activate the pairing of receptors (dimerization). This is required for many signaling pathways inside cells like human immune cells in order to trigger tailored responses.

“Our DNA-Origami team has advanced this bioinspired nanotechnology yet again by showing that they can be protected from degradation in vivo using a simply chemical coating approach, and that they can tune their update by designing their shape, size and compactness. This offers much greater and much finer control of design criteria necessary to accomplish goals of drug targeting that is possible with many other delivery systems,” says Donald Ingber, M.D., Ph.D., a co-author on one of these reports who is the Founding Director of the Wyss Institute, the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).

Other authors contributing to the DNA origami study are David Mooney, Ph.D., Core Faculty member of the Wyss Institute and the Robert P. Pinkas Family Professor of Bioengineering at SEAS, and past and present members of the Wyss Institute, including Frances M. Anastassacos,  Nandhini Ponnuswamy, Ph.D., Bhavik Nathwani, Ph.D.,  Leo Y.T Chou, Ph.D., Weiwei Aileen Li, Ph.D., Franziska G. Leifer, Ph.D., Chenxiang Lin, Ph.D., and Ju Hee Ryu, Ph.D. Wyss Imaging Core Manager Garry Cuneo; Mathias Vinter, Ph.D., who now is at the Centre for DNA Nanotechnology, Denmark.

This work was supported by Wyss Institute for Biologically Inspired Engineering at Harvard, an NIH New Innovator Award (no.1DP2OD004641-01), and an NSF Expeditions Award (no.CCF-1317291) to W.M.S. The work was further supported by the Intramural Research Program of KIST (no. 2E27960) to W.M.S. and J.H.R. M.M.C.B. was supported by the Human Frontier Science Foundation as a cross-disciplinary postdoctoral fellow. F.M.A. was supported by an Alexander S. Onassis Scholarship for Hellenes. N.P. was supported by the Schlumberger Faculty for Future Fellowship.

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