Fighting Tumors with DNA Origami

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The immune system works tirelessly to nip cancer cells in the bud, but occasionally a tumor wins and takes hold.1 To stop rogue tumor cells in their tracks, researchers are developing cancer vaccines that train immune cells to recognize tumor antigens. The success of these therapeutics tends to improve with the use of adjuvants, which are chemicals that kickstart the body’s defenses.2 However, throwing these molecules into the mix isn’t enough. Researchers must carefully manage the meeting between adjuvants and immune cells to produce an effective immune response.

In an article published in Nature Nanotechnology, scientists used DNA origami to tightly control the delivery of adjuvants to immune cells, and they developed this system into a cancer vaccine.3 They found that finetuning the distribution of these molecules across the surface of the DNA origami vaccine elevated tumor-fighting immunity, revealing that this technology may one day advance cancer therapies. 

Immune cells house receptors that detect common signs of pathogens, such as short DNA sequences called cytosine-phosphorothioate-guanine oligonucleotides (CpG) that were released from microbes, which trigger an immune cascade.4 Researchers previously found that multiple immune receptors converge when they sense crowds of CpG huddled over the immune cell surface, potentially influencing how intensely these receptors signal an immune attack on the incoming microbial DNA.5 Given CpG’s propensity to propel the immune system, vaccine developers have explored its prowess as an adjuvant.

Previously, vaccinologists decorated therapeutic nanoparticles with CpG adjuvants.6 However, William Shih, a synthetic biologist at Harvard University and an author of the Nature Nanotechnology paper, commented that the researchers couldn’t control the distribution of the short DNA molecules on the nanoparticle surface, which potentially led to suboptimal immune triggering. To precisely tune CpG spacing and spark the body’s defenses, Shih and his team explored the art of origami at the nanoscale.

“One of the attractive things about DNA origami is how relatively simple it is for anybody to design,” Shih noted. DNA bases strictly adhere to Watson-Crick base pairing, which makes it simple to simulate how multiple DNA strands will fold into a 3D structure.7 In fact, Shih remarked that it’s easy enough to design DNA origami models with nothing more than a pen and paper. “It’s a pretty unique property among materials,” he said.

Shih and his team folded DNA into cuboidal blocks by arranging multiple DNA origami structures side by side like a box of crayons. To transform the structure into a cancer vaccine, they attached tumor antigens to one end of the DNA molecules and CpG adjuvants to the other end. This wasn’t the first time that researchers combined CpG and DNA origami, but in previous iterations, they attached CpG along the length of the strands seven nanometers apart.By fusing the adjuvants to the ends of the double helices, Shih and his team could now draw them nearer, with a minimal distance of 2.5 nanometers. “Our paper was the first one to look at spacings down to this level,” Shih said.

To optimize their vaccine, which they named DoriVac, the team tested a variety of DNA origami blocks with different CpG spatial distributions. Each of them triggered helper T cells to adopt cancer-fighting properties. For example, they secreted antitumor cytokines, like tumor necrosis factor alpha (TNFα), a protein that triggers cancer cells to burst,  and they activated killer T cells, which target tumors for destruction.However, vaccines with adjuvants spaced 3.5 nanometers apart outperformed all other configurations at rewiring immune cells.

     Four designs of DNA origami with different patterns of CpG spacing.

Using DNA origami, researchers finetuned the spacing of CpG adjuvants (yellow) to maximize immune cell responses.

Wyss Institute at Harvard University

Once Shih and his team calibrated their DNA origami vaccine to maximize immunity, they put it to the test in mice. They vaccinated mice on days zero and seven before inoculating them with cancer cells at day 14. By day 28, all unvaccinated mice developed tumors compared to none of the vaccinated ones. To ensure that the vaccine’s 100 percent success rate was linked to adjuvant spacing, they gave another group of mice a control vaccine that contained free-floating CpG. This vaccine didn’t achieve a perfect score: 40 percent of the mice developed tumors, revealing the importance of CpG spacing.

Although the cancer vaccine worked well as a prophylactic, clinicians normally treat rather than prevent tumors in the clinic. To evaluate how well the vaccine works when cancers are already present, Shih and his team flipped the experiment and inoculated mice with tumor cells on day zero and vaccinated them on days three, seven and 14. However, the vaccine underperformed in this scenario: It only prolonged lifespan by a few days, and it didn’t wipe out any of the cancers. 

“My gut interpretation would be that once the tumor immune microenvironment is established, it’s much harder to change it,” said Meghan Morrissey, a cell biologist at the University of California, Santa Barbara, who was not involved with the study. She said that the tumor microenvironment can coax the immune system into becoming lenient, so the body’s defenses might need a jolt before they can be roped into battle.10

Shih considered jumpstarting immunity by combining DoriVac with another cancer therapeutic. “Immunotherapy is thought to be most effective when one brings many different modalities together,” he explained. Programmed cell death protein-1 (PD-1) is a common target for cancer therapy given the receptor’s role in shutting down T cells.11 Blocking PD-1 activity keeps T cells active, nudging them to thwart tumors. Pairing DoriVac with this immunotherapy sparked promising results. Tumors disappeared in every mouse, revealing that this therapeutic duo restored the vaccine’s potential.

Moving forward, Shih plans to test DoriVac’s safety and efficacy in humans. Morrissey argued that this technology could expand therapeutic research in other ways too. “I’m more excited about using DNA origami to figure out the design principles of the therapies, even if they don’t long-term end up being DNA origami,” she said. “DNA origami gives us a tool that’s both flexible and robust enough to ask questions that weren’t accessible before.”

Shih is an inventor on a US patent application for a DNA origami cancer vaccine (WO/2020/247724), and he is setting up a company to commercialize it.

References

1. Hanahan D. Hallmarks of cancer: new dimensionsCancer Discov. 2022;12(1):31-46.
2. Paston SJ, et al. Cancer vaccines, adjuvants, and delivery systemsFront Immunol. 2021;12:627932.
3. Zeng YC, et al. Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccinationNat Nanotechnol. 2024;9(3).
4. Salem AK, Weiner GJ. CpG oligonucleotides as immunotherapeutic adjuvants: innovative applications and delivery strategiesAdv Drug Deliv Rev. 2009;61(3):193-194. 
5. Schmidt NW, et al. Liquid-crystalline ordering of antimicrobial peptide–DNA complexes controls TLR9 activationNature Mater. 2015;14(7):696-700.
6. Klinman DM, et al. Use of nanoparticles to deliver immunomodulatory oligonucleotidesWIREs Nanomed Nanobiotechnol. 2016;8(4):631-637.
7. Loescher S, et al. 3D DNA origami nanoparticles: From basic design principles to emerging applications in soft matter and (bio-)nanosciencesAngew Chem Int Ed. 2018;57(33):10436-10448.
8. Comberlato A, et al. Spatially controlled activation of toll-like receptor 9 with DNA-based nanomaterialsNano Lett. 2022;22(6):2506-2513.
9. Carswell EA, et al. An endotoxin-induced serum factor that causes necrosis of tumorsProc Natl Acad Sci USA. 1975;72(9):3666-3670.
10. Enderling H, et al. Immunoediting: Evidence of the multifaceted role of the immune system in self-metastatic tumor growthTheor Biol Med Model. 2012;9(1):31.
11. Ohaegbulam KC, et al. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends in Molecular Medicine. 2015;21(1):24-33.

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