Design Considerations for Potent DNA-Platform-Based Cancer Vaccines

Abstract

Cancer vaccines have the potential to harness the strength of our immune system to fight cancer safely and effectively, but their potency relies on the presentation and tuned delivery of the vaccine’s components. Different than other delivery platforms, DNA-based nanostructures are highly programmable architectures that allow for precise control of size, geometry, flexibility, and spatial organization of the scaffold and conjugated moieties. The modularity of DNA-based nanostructures enables the field to consider and program various determinants of vaccine potency including biodistribution, cargo protection, cellular uptake, and intracellular processing. Consequently, numerous design strategies have emerged using various DNA-based materials. Collectively, these developments have illustrated how slight alterations in structure notably impacts the directions and strengths of the resulting immunostimulatory responses against tumors. As the relationship between structure and immunomodulation becomes increasingly understood, the potential of DNA-based nanostructures to serve as precise and potent cancer vaccine delivery platforms will be further realized.

Graphical Abstract

Introduction

Immunotherapies, which harness the strength of our immune system, have the potential to fight cancer with high tumor specificity, reduced side effects, and low recurrence rates. Cancer vaccines specifically comprise an immunostimulatory component (adjuvant) and target (antigen) and are designed to train immune cells to recognize and kill cancer. They are promising due to their targeted approach and ability to introduce immune memory. However, therapeutic cancer vaccines have not elicited wholly effective T cell responses and shown limited clinical success. This is in part due to ineffective delivery and presentation of vaccine components.¹

Vaccine delivery platforms can impact component processing and the resulting strength of the immune response through trafficking and protecting cargo, promoting cellular uptake, optimizing adjuvant presentation for elevated immunostimulation, and facilitating endosomal escape for antigen cross-presentation.^2,3

DNA-based nanostructures are a class of materials that leverage the ease of synthesis and self-assembling capabilities of nucleotides to form highly structured DNA architectures. They can be employed as vaccine component carriers for precise control of size, geometry, flexibility, and spacing.⁴ While DNA is also employed to encode antigenic proteins, this review will specifically cover the use of DNA as a biomaterial for vaccine component delivery. The modular nature of DNA-based nanostructures allows one to simultaneously tackle numerous shortcomings of current vaccines—biodistribution, cargo protection, cellular uptake, and intracellular processing—making them attractive for therapeutic development.⁴ Herein, we review current design considerations for DNA-based nanostructures for vaccine component delivery, and highlight DNA-based nanostructure strategies to address the design considerations and enhance vaccine potency.

Design Considerations

The design of DNA-based nanostructures as vaccine adjuvant and antigen carriers must consider the various drivers of potency for meaningful impact (Figure 1). Vaccine components should be trafficked to the lymph nodes, where dendritic (DCs) and T cells are colocalized.⁵ While nanocarriers between 20 - 100 nm are capable of circulating freely through the lymphatic system and may exhibit greater retention within lymph nodes, particles 100 nm or larger are more likely to be internalized by DCs at the injection site, and smaller particles (<20 nm) may exhibit accelerated clearance.⁶ Shape and aspect ratio affect the tissues in which nanocarriers accumulate and for how long.^5,7 Surface charge and composition dictate protein corona formation that modifies the pharmacokinetics and biodistribution of nanocarriers.⁸

Figure 1.

Design considerations for the development of potent DNA-based vaccine delivery platforms include biodistribution and lymph node drainage, protection from nuclease and protease degradation, uptake into antigen-presenting cells (APCs), and intracellular antigen and adjuvant processing.

Vaccine components are susceptible to degradation by nucleases and proteases in vivo. Therefore, nanostructure-mediated protection and prevention of premature cargo release before internalization by antigen-presenting cells (APCs) is essential. The stability of DNA-based nanostructures has previously been improved through modified oligonucleotides, functionalization with polymers, or encapsulation within protective coatings.⁹

Once near target cells, design characteristics can facilitate effective cellular uptake. For instance, DNA nanospheres are internalized more than both DNA nanorods and nanotiles of similar sizes.¹⁰ The size, geometry, flexibility, surface chemistry, and charge of DNA-based nanostructures determine the uptake mechanisms, which, in turn, affect intracellular processing.¹¹ The intracellular processing of delivered antigens, dependent on the nanocarrier, notably biases the immune response.¹² To elevate CD8⁺ T cell responses, strategies that enhance the endosomal escape of vaccine components are critical in the design of DNA nanostructures.²

Furthermore, the display of widely employed immunostimulatory components (e.g., CpG oligonucleotides) to endosomal toll-like receptors (TLRs) or other pathogen recognition receptors (PRRs) impacts the strength and direction of the immune response.^13,14 Multivalent and precisely-distanced display of CpG oligonucleotides biases towards humoral or cellular immunity, with the architecture of DNA nanostructures harnessed to tune TLR agonist presentation to induce potent anti-tumor responses.^14,15 In addition, the dynamic capabilities of DNA-based structures can promote TLR9 clustering and activation.¹⁶

Lastly, a key design decision for any cancer vaccine is the choice of antigen. DNA-based nanostructures can be used to protect antigen during trafficking and control the structural presentation of antigen to affect its cellular processing. When considering antigen selection, one may utilize shared tumor-associated antigens (TAAs) or patient-specific neoantigens. TAAs are overexpressed on tumor cells and can be pre-manufactured for broad applicability, but may suffer from central immune tolerance; patient-specific neoantigens arise from somatic mutations and are highly immunogenic and not subject to central tolerance, making them ideal for personalized vaccines despite requiring a more complex identification and manufacturing flow. Selection criteria also include antigen format (i.e., peptide, protein, mRNA-encoded), which influences how the antigen is processed and presented by APCs. These choices are not independent of the delivery platform; for example, the rigid architecture of DNA origami favors precise multivalent peptide display¹⁵, while the high cargo capacity of DNA nanogels is well-suited for protein or nucleic acid-based antigens.¹⁷ Thus, it is critical to consider the antigen design space and how it interacts with each DNA-based platform to select the most potent cancer vaccine for the intended application.

Sequence selection & molecular modifications

Designing the DNA sequence and whether it contains modifications is a critical first step in employing a DNA-based vaccine platform. While DNA can be intrinsically sensed by PRRs, the specific sequence and chemical modifications play a large role in how it is detected and processed by the cell. Chemical modifications to oligonucleotides can improve uptake, reduce nuclease degradation, strengthen target binding, and modulate stimulatory activity.¹⁸ Most modifications are made to either the backbone, nucleobase, or sugar (Figure 2),¹⁹ though they can be used in combination (e.g., gapmers, where modified blocks flank an unmodified “gap”; mixmers, where modified and unmodified nucleotides are mixed).¹⁸ The sequence and modifications dictate the receptor and ease through which DNA can be detected. Common receptors include TLR9, cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING), and Absent in Melanoma 2 (AIM2).

Figure 2.

Common oligonucleotide modifications to the backbone, sugar, and nucleobase (B = base, R = variable group). Common sugar modifications that alter the 2’-position of the pentose ring include 2′-fluoro (2′-F), 2′-O-methyl (2′-OMe), and 2′-O-methoxyethyl (2′-MOE). These modifications protect DNA from degradation and increase the specificity of modified strands for complementary DNA. 2′,4′-bridged nucleic acids (BNA) can also be used to modify the sugar by connecting the 2’ and 4’ ring positions. Examples include locked nucleic acids (LNA) and 2′-O,4′-C-ethylene bridged nucleic acids (ENA), which can be used to increase thermal stability and binding affinity. The DNA backbone can be modified to reduce nuclease degradation through the addition of a sulfur to replace the non-bridging phosphate oxygen forming a phosphorothioate. Lastly, the nucleoside DNA base can be modified to improve the stability of hybridized sequences. This is most often seen using the “5-methyl-C” modification, which introduces a methyl group to the cytosine base.

TLR9 is activated through the recognition of unmethylated CpG repeats in single stranded DNA. The location of these repeats is critical to activation. Removing the CpG sequence in the second and third position of TLR9 agonist “SY-ODN18,” reduced activation to ~10% of the original stand, whereas removing the 3’ CpG sequence in the tenth and eleventh position only mildly decreased activation to ~80% of the original.²⁰ Interestingly, the addition of a “5-methyl-C” modification—commonly used to increase the melting temperature and improve the stability of hybridized sequences—to the 5’ CpG motif decreased stimulation to ~20% of the unmethylated level, whereas methylating the 3’ CpG resulted in no statistical change.

The cGAS-STING pathway is responsible for sensing double-stranded DNA. Three base pair guanosine overhangs on self-hybridizing Y-form DNA have been shown to significantly improve cGAS activation compared to the same sequence without overhangs or with cytosine overhangs.²¹ Furthermore, a phosphorothioate (PS) backbone, which is widely used to reduce nuclease recognition and degradation,¹⁹ can impair cGAS binding, highlighting the different impact that modification can have based on the pathway targeted. A PS cGAS agonist, termed SVG3, induced 200-fold lower expression of Type I IFNs compared to its PO counterpart.²¹ However, other chemical modifications, such as locked nucleic acids (LNA), have been successfully utilized in cyclic dinucleotide (CDN) therapies to activate the cGAS-STING pathway more effectively. Endo-S-cGA_LMP, a synthetic LNA CDN, improved cGAS-STING activation in THP1 monocyte reporter cells compared to commonly used CDN 2’3’ cGAMP (EC₅₀ = 5.68 μM vs. 19.99 μM).²² LNA CDNs also enhanced stability in oxidative conditions and reduced enzymatic digestion.²²

The AIM2 pathway also senses double-stranded DNA and is typically considered to be non-sequence specific, as it recognizes the backbone and not a specific arrangement of bases. Due to this, Poly(dA:dT) is commonly used as an agonist; however some sequences, namely ODN TTAGGG (A151), which mimics telomeric DNA, strongly bind AIM2 and inhibit activation, highlighting the importance of sequence design even in widely-activated PRRs.²³ Moreover, its level of activation is dependent on DNA length, requiring 60 bp for minimal activation and >150 bp for optimal activation.²⁴

DNA sequence design and chemical modifications are vital to understand, as they impact the structural features of complex DNA nanostructures. Cube-like PS nucleic acid nanoparticles evaluated using solid-state nanopores were observed to be stiffer than their unmodified PO counterparts. The difference in electronegativity between sulfur and oxygen affected the bond angles in the DNA strands and resulting mechanical properties of the cubes.²⁵ Collectively, sequence design and modifications are integral because they influence stability, circulation, mechanics, immunogenicity, and binding both at the single strand level and at the level of more complex DNA-based platforms discussed in later sections.

Spherical Nucleic Acids

Spherical Nucleic Acids (SNAs), a class of oligonucleotide therapeutics comprising a nanoparticle core functionalized with a dense shell of radially arranged nucleic acids, offer a versatile platform for vaccine design.²⁶ This oligonucleotide presentation elevates uptake via multivalent interactions with class A scavenger receptors (SR-A), found on many cell surfaces, including APCs. Thus, SNAs efficiently deliver antigen and adjuvants to immune cells. This, combined with the shell’s conveyed protection from nuclease degradation, enhances SNAs’ overall immunomodulatory capabilities compared to a subunit vaccine admix,²⁶ and immunostimulatory SNA structures have led to promising clinical trials.²⁷ By harnessing aforementioned chemical oligonucleotide modifications with the SNA, further improvements on nuclease resistance and half-life can be achieved.²⁸

SNAs allow for the combination of antigen and adjuvant and/or multiple adjuvants or antigens in a structurally-controlled manner, which improves synergistic effects.^29,30 To explore immunological effects of adjuvant and antigen co-presentation, HPV16 E7_11-19 peptide antigen and CpG adjuvant were combined in three SNAs with different structural arrangements: CpG adjuvant was radially attached to the liposome core while antigen was either (1) inside the core (E-SNA), (2) hybridized by conjugation to complementary CpG strands via a cysteine residue that was added to the N terminus of the peptide (N-HSNA), (3) hybridized by conjugation to complementary CpG strands via a cysteine residue that was added to the C terminus of the peptide (C-HSNA).³¹ Although composition was nearly identical, N-HSNA elicited the strongest response, inducing ~8-fold higher interferon-γ secretion and ~2.5-fold greater cytotoxicity. This highlights the impact of structured antigen presentation on downstream immunity.

The release kinetics of multiple vaccine components can be controlled through enzyme-cleavable linkers,³² reducible or non-reducible linkers,³³ and modulating anchoring chemistry between the oligonucleotide and nanoparticle core.³⁴ Altering the release kinetics of antigen or adjuvant importantly affects processing and presentation and biases the immune response. An endoplasmic reticulum aminopeptidase1 (ERAP1)-responsive linker was used to tune antigen processing efficiency.³⁵ ERAP1 is a protease in the endoplasmic reticulum involved in generating major histocompatibility complex class I (MHC I) epitopes. SNAs where antigen was attached to the nanoparticle through a Leu-Ala-Met-Met ERAP1-responsive linker exhibited a 30% increase in surface antigen presentation and a 5-fold increase in CD8⁺ T cell proliferation compared to a different ERAP1-responsive linker (Leu-Ala-Lys-Lys) with less efficient cleavage.

SNAs’ modular design characteristics can be applied in various ways to enhance their delivery and immunomodulatory capabilities, making them a promising approach for next-generation vaccines and immunotherapies (Table 1).

Table 1.

Overview of characteristics of current DNA-platform-based cancer vaccines, arranged in order of discussion in this review.

	SNAs	DNA Origami	DNA nanogels/Hydrogels	Polyhedral DNA	Branched DNA	DNA Nanoflowers
Structure	Nanoparticle core with a dense shell of radially-oriented nucleic acids; particle size range of 10-250 nm dependent on core and DNA length	Highly defined 2D/3D; precise ligand placement (e.g., 3.5 nm spacing), particle size range of 10-100nm	3D polymer networks; macroscopic depots (mm to cm scale) or <200 nm nanogels	Simple 3D geometries; precise peptide placement, particle size range of 10–150 nm	Covalently conjugated DNA in stem-and-branch configurations, particle size range of 5–20 nm	Dense assembly of single-stranded DNA particle size range of 200–300 nm
Bio-distribution	Dense shell prolongs circulation and enhances targeting	Tuned by size, shape, and surface ligands (e.g., aptamers) to enhance accumulation in target tissues and prolong circulation	Injectable depot for local retention; nanogels for lymph node trafficking	Tuned by particle size, geometry, structural rigidity, and surface modifications to enhance accumulation in target tissues and prolong circulation	Multivalent DNA arrangement prolongs circulation and enhances targeting	Tuned by particle size, geometry, structural rigidity, and surface modification to enhance accumulation in target tissues and prolong circulation
Protection	Dense shell enhances nuclease resistance	Coatings (e.g., oligolysine-PEG, virus capsids) or structural design shield cargo from degradation	Physical encapsulation within gel matrix shields cargo from degradation	Tuned geometric structures enhance nuclease protection	Multivalent DNA arrangement enhances nuclease resistance	Dense structure efficiently delivers nucleic acids and protects from nucleases
Cellular Uptake	Dense shell encourages uptake through SR-A	Cell-type & structure-dependent; efficient DC uptake reported; endocytosis (SR-A mediated)	Can be designed for efficient uptake (e.g., with DC-targeting aptamers)	Efficient endocytosis Reported; no clear mechanism known	Multivalent DNA arrangement encourages uptake through SR-A	Efficient endocytosis Reported; no clear mechanism known
Intra-cellular Processing	Tuned by stimuli-responsive linkers (e.g., ERAP-responsive) for enhanced antigen presentation	Tuned by stimuli-responsive locks (e.g., pH, redox, enzyme, light) for controlled release	Tuned by TME-responsive moieties (e.g., pH-sensitive i-motif) for dissociation; acid-labile linkers for traceless release of unmodified cargo	Tuned by size (e.g., side length) and antigen spacing for enhanced antigen presentation	Tuned by valency, antigen: adjuvant ratios, and antigen placement for enhanced antigen presentation	Tuned by stimuli-responsive linkers for controlled release
Potency	8-fold higher IFN-γ secretion, 2.5-fold greater cytotoxicity via defined structural presentation	Potent Th1 responses via defined spacing; boosts B cell & GC responses	High capacity for multi-agent co-delivery; induces ICD & potent T-cell (CD8+ CTL) responses	Induces prophylactic antitumor immunity; higher IgG titers via defined antigen spacing	30% higher tumor cell killing via defined structural presentation	High cargo capacity; adjuvant delivery reduces metastasis compared to free CpG
Clinical Readiness	Clinical; TLR9-agonist was in Phase 1b/2 trials for patients with advanced solid tumors	Preclinical; early human trials emerging; hindered by stability & scalability	Preclinical; hindered by scalability & reproducibility	Preclinical; balance between programmability and scalability; hindered by folding efficiency	Preclinical; balance between programmability and scalability; hindered by low synthetic yields	Preclinical; more cancer vaccine-specific research necessary
References	[26],[27],[28],[29],[30],[31],[32],[33],[34],[35]	[15],[36],[37],[38]	[17],[39],[40]	[41],[42]	[43],[44],[45]	[46],[47]

DNA origami

DNA origami structures DNA into complex, nanometer-scale forms through hybridization, and enables precise control over the spatial arrangement of molecules on its surface. Like SNAs, DNA origami leverages the programmability of DNA for vaccine construction, generating highly defined, rigid nanostructures with unique spatial precision (Table 1). The precise design of adjuvant spacing and antigen positioning enables structural feature optimization and directly governs immunological outcomes.¹⁵ Among CpG spacings tested (2.5, 3.5, 5.0, and 7.0 nm), the 3.5 nm interval induced the strongest Th1 immune response, including CD8⁺ T, Th1-CD4⁺ T, and NK cells. This was likely favored as the spacing mimics the natural dimerization distance of TLR9 on immune cells.¹⁵

DNA origami also enables simultaneous co-delivery of antigen and adjuvant, where the two components can be independently attached to distinct faces of the nanostructure at a density of up to 150 molecules per origami.¹⁵ This precise spatial control enables antigen-focused germinal center responses by minimizing off-target B cell activation against the scaffold itself—a common limitation of protein-based nanoparticles where up to 70% of the antibody response is against the carrier—thereby directing the immune system to mount a potent and specific response against the tumor antigen.³⁶ In a humanized mouse model, DNA origami vaccines expanded 3-fold more antigen-specific germinal center B cells over a benchmark protein nanoparticle displaying the same antigen, demonstrating that the DNA origami scaffold’s inert nature avoids the off-target immune responses often generated against protein carriers.³⁶ This platform could be adapted for personalized vaccines where patient-specific neoantigens can be precisely arrayed alongside optimized adjuvants, thus enabling modular vaccine design tailored to an individual’s tumor mutational profile.

DNA origami has the potential to overcome trafficking and biodistribution challenges by tuning the size and shape or by attaching targeting moieties to the surface (i.e., DNA aptamer to target cancer or immune cells) to alter biodistribution.³⁷ For instance, one study systematically compared 6-helix bundles (~6nm diameter) versus 24-helix (~14nm) and found that differences in size and compactness influenced cellular uptake and biodistribution profiles.³⁸ Protective coatings placed around the origami core can also shield the structure from nuclease degradation, prevent premature breakdown by serum enzymes, and extend its half-life and stability in vivo (e.g., >80% of coated origami remains intact after 60 min in DNase I at a high enzymatic activity level (50 Kunitz units/mL), compared to complete degradation of uncoated structures).³⁸ Moreover, the ability to control antigen display density on the surface (as in “DNA Virus-like particles”) can engage humoral immunity more effectively, expanding 3-fold more antigen-specific germinal center B cells and priming highly epitope-specific responses compared to clinical protein nanoparticles displaying the same HIV immunogen.³⁶

Efforts to overcome the endosomal barrier with DNA origami have been pursued. Researchers generated multi-step, stimuli-responsive designs that encapsulate and protect cargo during circulation, but upon reaching the cell, are triggered by the high levels of glutathione in the cytoplasm environment to release cargo through unrolling of the tubular structure.³⁷

DNA origami incorporates key design considerations for cancer vaccines by enabling precise spatial control over component arrangement, size, shape, and surface modifications. These capabilities position it as a versatile platform for precision cancer immunotherapy.

DNA nanogels and hydrogels

DNA-based hydrogel systems offer distinct solutions by leveraging their programmable three-dimensional networks. DNA nanogels function as discrete, high-capacity carriers that protect encapsulated payloads from degradation while enabling stimuli-responsive disassembly and traceless release upon cellular uptake.^17,39 At the macroscopic scale, injectable DNA hydrogels serve as in situ vaccine depots which sustain the release of immunogenic cell death (ICD)-inducing adjuvants and personalized antigens from tumor cells.⁴⁰

DNA nanogels can function as modular scaffolds for co-delivery of multiple payloads, which is a principle shared across DNA-based platforms, including SNAs and DNA origami, which achieve spatial control on rigid nanostructures using multi-agent functionalization.^15,37 However, DNA nanogels are distinctly advantageous due to their three-dimensional porous network, enabling high encapsulation efficiency (e.g., up to 80-90% loading of therapeutic nucleic acids) and environmentally-triggered release.³⁹ A ‘three-in-one’ nucleic acid hydrogel, assembled through crosslinking programmed death-ligand 1 (PD-L1) siRNA with a Y-shaped motif containing both SN38 and CpG adjuvant,⁴⁰ achieved co-delivery of three functional agents: chemotherapeutic drug to induce ICD, immune agonist, and immune checkpoint inhibitor to reduce immunosuppression. A DNA-polyacrylamide nanogel cancer vaccine (DPCV) uses a similar mechanism, assembling a DNA nanogel scaffold with CpG adjuvant, tumor-targeting aptamers, and chemotherapeutics.¹⁷ These gel matrices physically protect payloads from degradation (e.g., >80% encapsulated DNA remains intact after 24 h in serum) and extend the half-life and bioavailability in vivo.¹⁷

Nanogels leverage environmentally sensitive moieties to control dissociation and undergo structural collapse or disintegration upon stimulus, which can be tuned to occur in the tumor microenvironment (TME). The DPCV platform contains i-motif DNA structures that change conformation in acidic conditions (pH<6.5) and trigger dissociation in cellular endosomes at the tumor site.¹⁷ Nanogels also allow ‘traceless’ release of adjuvants and antigens in their original, unmodified forms to preserve bioactivity via acid-labile chemical linkers triggered in endosomes.³⁹

DNA nanogels can function as a therapeutic depot, inducing ICD to release tumor-associated antigens in situ. This enhances patient-specific antigen presentation, which further promotes DC maturation (>70% CD80/86 upregulation) and CD8⁺ T cell infiltration (3- to 5- fold increase in intratumoral CD8+ T cells).⁴⁰ Combining therapeutic depot nanogels with PDL1-targeting siRNA simultaneously reduced immunosuppression and enhanced TME immunogenicity.⁴⁰ In mouse models of melanoma, breast, and pancreatic cancer, this approach reduced tumor growth by >70%.^17,40

Together, these nanogel and hydrogel platforms address critical design challenges, including cargo protection, controlled release kinetics, and the coordination of multi-agent delivery, by exploiting unique programmability and environment responsiveness (Table 1).

Other DNA-based platforms and hybrid platforms

Other DNA-based architectures and hybrid platforms have also emerged, which incorporate key design considerations to enhance overall vaccine performance (Table 1). These include tetrahedral and other polyhedral DNA frameworks, DNA dendrons and polypod DNA assemblies, and rolling circle amplification (RCA) derived nanostructures (i.e., DNA nanoflowers).

Tetrahedral and other polyhedral DNA frameworks are discrete, closed three-dimensional nanostructures assembled from oligonucleotides into defined geometries. Unlike DNA origami, which typically relies on scaffold-guided folding to generate complex nanostructures, polyhedral DNA frameworks are generally constructed from a smaller number of strands and represent a structurally simpler class of DNA-based materials. Their defined three-dimensional architectures via simple self-assembly make them beneficial for organized presentation and delivery of vaccine components. A CpG-loaded tetrahedral framework nucleic acid (CpG-tFNA) showed excellent cellular permeability and, through electrostatic interactions, enabled co-delivery of positively-charged peptide adjuvant β-defensin 2 and antigen-encoding mRNA.⁴¹ This system promoted antigen expression and presentation by DCs and induced prophylactic antitumor immunity. Researchers also engineered various tFNAs with controlled rigidity and size to precisely organize epitopes and showed that peptide epitopes displayed on tFNAs with a side length of 37 base pairs (epitope spacing of ~12.58 nm) elicited higher IgG titers.⁴²

DNA dendrons and polypod DNA assemblies comprise multiple arms that form multivalent, branched architectures. Their modular designs enable spatial organization of immunostimulatory motifs and tuning of motif stoichiometry, placement, and structure to uncover key structure-function relationships. Researchers designed four different polypod DNA structures containing CpG motifs at different positions and showed that CpG placement in the 5′-overhang single-stranded region was more effectively recognized by TLR9 than those in the double-stranded region.⁴³ DNA dendrons simplify the structural architecture of SNAs by radially presenting covalently-conjugated DNA in a stem-and-branch configuration while retaining similar pharmacokinetic advantages (i.e., enhanced uptake via SR-A, prolonged circulation, reduced degradation).^44,45 Moreover, DNA dendrons enable studies on the impact of antigen and adjuvant placement and valency with molecular precision. Indeed, 6 branch DNA dendrons containing a single CpG sequence in the stem, compared to placement in the branches, were most effective at delivering adjuvant, whereas antigen placement was less critical than overall valency in driving antitumor potency. Increased antigen loading enhanced uptake and immune activation (an antigen/adjuvant ratio of 6:1 versus 1:1), highlighting the importance of structurally-defined presentation in shaping vaccine efficacy.⁴⁵

Rolling circle amplification (RCA) generates ultralong repetitive single-stranded DNA that can be further organized into defined nanoassemblies through self-assembly or condensation. This dense organization supports high cargo incorporation, tandem motif presentation, and structural stability to deliver adjuvants and antigens. RCA-generated DNA nanoflowers efficiently delivered CpG motifs protected from nuclease degradation, and promoted macrophage polarization and secretion of immunostimulatory cytokines.⁴⁶ Researchers also developed an RCA-based DNA nanococoon loaded with CpG motifs and anti-PD-1, which enabled MMP-9-triggered release of an encapsulated endonuclease to fragment the nanococoon and release both cargos, resulting in greater therapeutic efficacy against B16-F10 metastasis than free CpG and anti-PD-1.⁴⁷

Hybrid platforms, including DNA–mRNA, DNA–polymer, and DNA–metal systems, expand the functional scope of DNA-based cancer vaccines by combining DNA programmability with complementary advantages of other materials. A representative DNA–mRNA hybrid system uses a multimodule DNA nanostructure to enhance mRNA vaccine efficacy.⁴⁸ It incorporates poly(T) mRNA-anchoring sequences, CpG, acidic pH-responsive C-rich sequences, and DC-targeting aptamers through a two-step hybridization chain reaction of three DNA hairpins. It forms micro-sized aggregates in acidic lysosomes to enhance CpG activity, then escapes into the cytoplasm to promote localized mRNA translation, resulting in potent antitumor immunity and inhibited tumor growth and metastases. In DNA–polymer hybrids, polymers provide structural support, cargo protection, and delivery capacity. CpG-containing DNA hairpins have been used as crosslinkers within polymeric networks, simultaneously enabling stimulus-responsive assembly and immunostimulation.⁴⁹ Separately, a DNA scaffold was integrated with PLGA nanoparticles to precisely control the relative presentation of DEC205- and Clec9A-targeting antibodies, thereby promoting preferential delivery of STING agonist MSA-2 to cDC1s and enhancing CD8⁺ T-cell-mediated antitumor immunity.⁵⁰ DNA–metal hybrids can potentiate innate immunity through nanoscale structure. Mn²⁺ enhances CpG-mediated activation through the STING–NF-κB axis, which led to the development of Mn-containing CpG nanoadjuvants.⁵¹ Recently, researchers employed a Mn-responsive DNA system to form coordination nanofibers in situ that promote lysosomal escape and elicit DC activation, antigen-specific CD8⁺ T-cell responses, and durable immune memory.⁵² Collectively, these hybrid platforms illustrate how DNA scaffolds can serve as a modular framework for integrating antigen expression, adjuvant activity, and delivery control with complementary advantages of other materials.

Translational challenges and clinical outlook

Although DNA-platform-based cancer vaccines remain at an early stage of translation, the broader field of oligonucleotide therapeutics has already established a strong clinical foundation for nucleic acid medicines. Multiple chemically-modified antisense oligonucleotides, such as Nusinersen, Inotersen, and Volanesorsen, have received regulatory approval, confirming that chemically defined nucleic acid therapeutics can be manufactured under GMP conditions.⁵³ Some DNA-based nanostructures have entered early clinical evaluation. Notably, cavrotolimod (AST-008), a TLR9-agonist SNA, has undergone phase 1b/2 clinical studies in patients with advanced solid tumors (i.e., Merkel cell carcinoma, cutaneous squamous cell carcinoma), illustrating that DNA-based platforms can progress toward clinical application.²⁷

Compared with simple oligonucleotide therapeutics, however, multicomponent DNA nanostructures introduce additional challenges in chemistry, manufacturing and controls, because the final product is defined not only by nucleotide sequence but also by supramolecular assembly and structural integrity. For example, the translation of DNA origami is currently constrained by the need for long scaffold strands, numerous unique staple strands, high folding efficiency, extensive purification, endotoxin control, batch-to-batch reproducibility, and high manufacturing costs. Furthermore, architecture-dependent pharmacokinetics, biodistribution, and structural stability remain insufficiently characterized for many DNA nanomaterials. In this context, simpler architectures such as tetrahedral DNA nanostructures, branched DNA assemblies, and DNA dendrons have the potential to provide a more practical balance between structural programmability and manufacturability. The translational niche of DNA nanomaterials also differs from that of established vaccine platforms such as messenger RNA encapsulated in LNPs. These nanoparticle systems provide highly efficient nucleic acid delivery and benefit from substantial clinical and manufacturing maturity. Polymer nanoparticles offer broad formulation flexibility. Virus-like particles enable highly repetitive antigen presentation. However, DNA-based platforms are uniquely advantageous as precise molecular organization has been shown to contribute directly to biological function. Their sequence-defined architecture enables independent control over geometry, valency, stoichiometry, and nanoscale spacing, allowing rational regulation of receptor clustering, antigen–adjuvant co-display, and coordinated immune activation. Future clinical translation will therefore depend on integrating structural programmability with manufacturability and therapeutic need, favoring the simplest architecture capable of achieving the desired complex biological function.

Conclusion and Perspective

Herein, we highlight the needed design considerations to generate the most potent cancer vaccines, as well as strategies to implement them using DNA-based nanostructures. Across these platforms, structural features such as size, geometry, flexibility, molecular spacing, and surface organization are active determinants of biodistribution, payload protection, cellular uptake, intracellular processing, and ultimately immunological outcomes. Various DNA platforms provide distinct design options to drive these features in a programmable way, with individual systems particularly suited for precise cargo organization, multivalent ligand display, controlled delivery, or multifunctional integration with complementary materials. However, the full potential of these platforms has not yet been realized, as important challenges remain in balancing structural sophistication with scalability, stability, and practical applicability. Future development of DNA-platform-based cancer vaccines must systematically explore how structural features dictate immunological function, leveraging new chemical advances to overcome delivery, processing, and immune propagation challenges. As these structure-function relationships become better defined and engineering solutions mature, DNA-based platforms will be uniquely positioned to design precision immunity against aggressive and evasive cancers.