DNA barrel-shaped nanostructure vaccine

Abstract

The present disclosure relates to nucleic acid-based vaccines for cancer and other diseases.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 414,380, filed Oct. 7, 2022, and U.S. Provisional Application No. 63 / 489,561, filed Mar. 10, 2023, under 35 U.S.C. § 119(e), the entire contents of each of which are hereby incorporated by reference.

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (H049870783WO00-SEQ-MSB.xml; size: 11,734 bytes; and creation date: Oct. 2, 2023) are hereby incorporated by reference in their entirety. Background of the Invention

[0005] Neoantigens arise from non-synonymous mutations in cancer genomes and are not subject to central tolerance, making them potentially immunogenic. Vaccination against tumor-specific neoantigens minimizes the potential induction of central and peripheral tolerance and the risk of autoimmunity. Neoantigen-based cancer vaccines have recently shown significant therapeutic potential in both preclinical and early clinical studies. For example, in situ vaccination utilizes the entire antigen repertoire of the tumor to minimize immune escape, overcoming the limitations of conventional therapeutic cancer vaccines for melanoma and other cancers. Individualized cancer vaccination using nanomaterials to capture antigens ex vivo also holds great promise for cancer immunotherapy. Summary of the Invention

[0007] To date, the therapeutic responses to neoantigen vaccines have revealed limited cytotoxic CD8 + T cells but a high frequency of CD4 + T cells. An effective vaccine strategy, such as the one provided herein, targets neoantigens, which can minimize the risk of autoimmune responses that may be potentially caused by whole tumor cell or lysate vaccine approaches. Few studies have provided viable methods for vaccine strategies that capture neoantigens. In some cases, effective vaccination relies on the administration of immune adjuvants to activate antigen-presenting cells (APCs). For example, CpG oligodeoxynucleotides (CpG ODNs) are common adjuvants for the pattern recognition receptor TLR9 and are known to polarize anti-tumor immune responses. Studies have shown that both the spacing and density of nanoscale CpGs play important roles in polarizing Th1 or Th2-type immune responses. In lymphoma patients, vaccination with CpG in combination with local radiotherapy has also led to CD8 +The T cell proliferation level is relatively low. Understanding the role of adjuvant spacing and therapeutically exploiting spatial distribution to control immune responses has been technically difficult because the materials currently available for presenting these ligands can generally only control the average spacing and cannot provide precise and uniform spacing.

[0008] DNA origami (e.g., organizing DNA into three-dimensional structures) can offer all the advantages associated with nanoparticles for vaccination. Moreover, DNA origami can uniquely enable the study of the impact of spatial control on ligands and the co-delivery of antigens and adjuvants to APCs. It is hypothesized that the hydrophobicity of peptides is a major influencing factor in damage-associated molecular pattern (DAMP)-related signaling. DNA origami can construct structures with hydrophobic motifs for capturing neoantigens, which depends on the hydrophobicity of immunogenic neoantigens. For example, a neoantigen capture motif can be constructed within a DNA origami structure shaped like a barrel (referred to herein simply as a "barrel") to avoid aggregation during engineering. In some embodiments, adjuvants can be constructed on the surface of the barrel, thus ensuring the optimal spacing required for dendritic cell stimulation and Th1-type polarized immune responses. In the present disclosure, barrels with optimal adjuvant (e.g., CpG and / or dsRNA) spacing are constructed, and neoantigen capture motifs are constructed within the barrels. This nucleic acid origami vaccine platform (also known as DoriVac) is capable of co-presenting the captured neoantigens and adjuvants with optimal spacing to APCs to obtain a neoantigen-specific Th1-type polarized immune response and enhance cancer treatment efficacy. Specifically, in some embodiments, the DNA origami vaccine comprises a nucleic acid barrel-shaped nanostructure conjugated with adjuvant molecules and antigen capture motifs. In some embodiments, the DNA origami vaccine comprises a nucleic acid barrel-shaped nanostructure conjugated with antigen molecules and adjuvant molecules. Additionally, in some embodiments, this DNA origami vaccine platform is capable of co-presenting the captured neoantigens and adjuvants with optimal spacing to APCs, either systemically or in situ.

[0009] The vaccine platform provided herein is highly translatable. The immunogenicity of DNA origami itself is limited. Other components used herein (e.g., adjuvants such as CpG and double-stranded RNA (dsRNA), and / or antigen capture motif classes such as coiled-coil peptides) have been previously used in clinical settings. This DNA origami vaccine platform can be used for many cancer types and in combination with other chemotherapeutic or immunotherapeutic agents. Additionally, this DNA origami vaccine platform can also be used for vaccination against other diseases (e.g., infectious diseases). Brief Description of the Drawings

[0011] Figure 1 : Basic barrel structure design. The DNA double helix is arranged in three layers and bent to fold into a closed barrel-shaped structure.

[0012] Figures 2A - 2B : Design of DNA Origami Barrels for Payload Construction. (A) Schematic showing the modification positions of the double helix for different payloads. The double helix was numbered in the CaDNAno software program for reference. (B) Handle region modification sites shown by CaDNAno DNA routing planning. Pink: Cy5, Green: CpG, Purple: dsRNA, Blue: CCP.

[0013] Figures 3A - 3D : Optimization of Barrel Structure Construction. (A) Optimization of the folding conditions of the barrel structure. (B) TEM images at low and high magnifications showing the monodispersity and robustness of the barrel structure produced. (C) Optimization of purification using different concentrations of PEG buffer. (D) Folding of the barrel structure using different excess amounts of CpG and dsRNA.

[0014] Figures 4A - 4D : DNA Origami Barrels Constructed Using dsRNA and CpG. (A) TEM image and model image of the barrel structure. Dimensions of the barrel structure: 30 x 60 nm. (B) Modification sites of Cy5, dsRNA, and CpG on the outer surface of the barrel. (C) Modification sites of the coiled-coil motif inside the barrel. (D) Conjugation of different excess concentrations of CpG and dsRNA (25 nt) to the barrel. Conjugation was successful.

[0015] Figures 5A - 5B : Barrels Constructed Using CpG and dsRNA Stimulated HEK blue Cells. (A) HEK blue detection of TLR9-expressing cells activated by different barrel structures and controls. (B) HEK blue detection of TLR3-expressing cells activated by different barrel structures and controls.

[0016] Figures 6A - 6G: Coiled - coil peptides (CCPs) can be successfully constructed in barrels and exhibit self - trapping. (A) Amino acid sequences, DNA attachment positions, and net charges of the peptides used in this study (azK represents azido - lysine). (B) Denaturing PAGE gel results show that PBS and 5 - fold excess of the peptide are suitable conditions for CCP - oligonucleotide conjugation. (C) Pure peptide - DNA conjugates are obtained by PAGE purification, as shown by a single band. From left to right, the amount of product loaded per well gradually increases. (D) All 5 peptides are successfully conjugated to the barrel through handle / anti - handle DNA hybridization. (E) TEM images of the barrel under different conditions. After conjugating CCP - E4_N (right panel), compared with the barrel (left panel) and the barrel with CpG and dsRNA (left and middle panels), a reduction in the blank area (white space) inside the barrel is observed. Dimensions of the barrel: 30x60 nm. (F) Agarose gel results show that barrels are constructed with different excesses of CCP. B - C - R: barrel - CpG - dsRNA. 1xP means incubating the CCP without the anti - handle region with the barrel at an equimolar ratio. (G) After DNase digestion, the peptide conjugation efficiency is determined by silver staining of the SDS PAGE gel. The increase in peptide intensity confirms that once CCP is constructed inside the barrel, more CCP is self - trapped.

[0017] Figures 7A - 7C : Barrel - based vaccine platform captures short peptides. (A) Silver staining of SDS PAGE gel shows that after digesting all barrel structures with DNase, CCP captures hydrophobic peptides. Upper band: DNase I. Lower band: CCP + short peptide (hydrophobicity: FGFGF > RGFGY > GGFGG). (B) Quantification of band intensity. (C) HPLC results show the peptides remaining in the supernatant after precipitating barrel - based vaccine nanoparticles. The less shown in the histogram, the more peptides are captured by the barrel - based vaccine platform.

[0018] Figures 8A - 8D : Barrel - based vaccine platform captures proteins released from irradiated tumor cells. (A) Schematic diagram shows how cells are irradiated and how the barrel - based vaccine platform captures and analyzes proteins. (B, C) Images show CT26 and B16F10 cells before and after irradiation. (D) Silver staining results show the captured proteins running through a 4 - 12% SDS PAGE gel.

[0019] Figures 9A - 9F : Mass spectrometry analysis of the captured proteins. (A - D) Preliminary tests are performed using mass spectrometry to check the antigens captured by the barrel, which may be related to improved vaccination. (E - F) Mass spectrometry studies compare the amounts of antigens captured under different barrel conditions.

[0020] Figures 10A - 10D: Efficacy of Barrel - shaped DNA Origami Vaccine (DoriVac). (A) Establishment of B16F10 tumor model and treatment protocol. (B) B16F10 tumor images at day 14 after 3 doses of vaccination and 5 doses of anti - PD - L1 (if applied). (C) Mouse tumor growth curve (n = 7). (D) Mouse survival curve (n = 7).

[0021] Figures 11A - 11F : Immune cell profiling. (A, B) Analysis of the expression levels of CD40 and PD - L1 on CD11c - positive dendritic cells in the draining lymph nodes by flow cytometry. (C, D) Analysis of the expression level of CD69 on CD4 - and CD8 - positive T cells in the draining lymph nodes by flow cytometry. (E, F) Analysis of the expression level of IFNγ on CD4 - and CD8 - positive T cells in the draining lymph nodes by flow cytometry.

[0022] Figures 12A - 12E: Efficacy of Barrel - shaped DoriVac with fewer B16F10 cell inoculations. (A) Establishment of B16F10 tumor model and treatment protocol. (B) B16F10 tumor images at day 17 after 3 doses of vaccination and 5 doses of anti - PD - L1 (if applicable). (C) Mouse tumor growth curve (n = 7). (D) Mouse survival curve (n = 7). (E) Survival curve of mice after tumor rechallenge of surviving mice from (D) with 1x10 5 B16F10 tumor cells. The control was naïve mice that received the same number of cells.

[0023] Figures 13A - 13D: Efficacy of Barrel - shaped DoriVac for in - situ vaccination. (A) Establishment of B16F10 tumor model and treatment protocol. Intramural application of doxorubicin (4 mg / kg) to induce local immunogenic cell death. Intramural application of Barrel - shaped DoriVac (without prior antigen capture), and anti - PD - L1 was administered around the tumor tissue. (B) B16F10 tumor images at day 12 after 1 dose of vaccination and 3 doses of anti - PD - L1 (if applied). (C) Mouse survival curve (n = 7). (D) Survival curve of mice after tumor rechallenge of surviving mice from (C) with 1x10 5 B16F10 tumor cells. Control mice were naïve mice that received the same number of cells.

[0024] Figures 14A - 14D: Efficacy of Barrel - shaped DoriVac in MC38 colon cancer model. (A) Establishment of MC38 tumor model and treatment protocol. (B) MC38 tumor images at day 15 after 3 doses of vaccination and 5 doses of anti - PD - L1 (if applied). (C) Mouse survival curve (n = 7). (D) With 2x105 Survival curves of mice after tumor rechallenge with live MC38 tumor cells in (C). Controls were naive mice that received the same number of cells.

[0025] Figures 15A - 15B : Prophylactic vaccination. (A) C57BL6 mice received two doses of vaccine on day 0 and day 7. On day 7, 1x10 5 B16F10 cells were inoculated into the mice. The survival of the mice was recorded (n = 5). (B) C57BL6 mice received two doses of vaccine on day 0 and day 7. On day 7, 2x10 5 MC38 cells were inoculated into the mice. The survival of the mice was recorded (n = 5). DETAILED DESCRIPTION OF THE INVENTION

[0027] Provided herein are nucleic acid nanostructure vaccines that comprise a nucleic acid nanostructure (e.g., a nucleic acid barrel nanostructure) linked (e.g., conjugated) to an adjuvant molecule, an antigen molecule, and / or an antigen capture motif. In some embodiments, provided herein are nucleic acid barrel nanostructures conjugated to an adjuvant molecule and an antigen molecule. Further embodiments provide nucleic acid barrel nanostructures conjugated to an adjuvant molecule and an antigen capture motif (e.g., a coiled coil peptide (CCP)).

[0028] In some embodiments, the nucleic acid nanostructure comprises at least 5, at least 10, or at least 20 adjuvant (e.g., CpG and / or double-stranded RNA (dsRNA)) molecules per type. In some embodiments, the nucleic acid nanostructure comprises at least 5 (e.g., CpG and / or dsRNA) molecules. For example, the nucleic acid nanostructure can comprise 5 - 200, 5 - 175, 5 - 150, 5 - 125, 5 - 100, 5 - 85, 5 - 75, 5 - 65, 5 - 55, 5 - 50, 5 - 45, 5 - 40, 5 - 35, 5 - 30, 5 - 25, 5 - 20, 10 - 55, 10 - 50, 10 - 45, 10 - 40, 10 - 35, 10 - 30, 10 - 25, 10 - 20, 15 - 55, 15 - 50, 15 - 45, 15 - 40, 15 - 35, 15 - 30, 15 - 25, or 15 - 20 adjuvant (e.g., CpG and / or dsRNA) molecules. In some embodiments, the nucleic acid nanostructure comprises 100 to 200, 150 to 200, 100 to 150, 50 to 100, 25 to 75, 25 to 50, 5 to 25, 10 to 25, or 15 to 25 adjuvant (e.g., CpG and / or dsRNA) molecules. In some embodiments, the nucleic acid nanostructure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 adjuvant (e.g., CpG and / or dsRNA) molecules per type. In some embodiments, the nucleic acid nanostructure comprises 18 adjuvant (e.g., CpG and / or dsRNA) molecules per type.

[0029] In some embodiments, each adjuvant molecule among the plurality of adjuvant molecules is evenly spaced 4.0 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, or 7 nm from any other adjacent adjuvant molecule. In some embodiments, each adjuvant molecule among the plurality of adjuvant molecules is evenly spaced 4.3 nm, 4 nm - 10 nm, 4.5 nm - 10 nm, 5 nm - 10 nm, 5.5 nm - 10 nm, 6 nm - 10 nm, 6.5 nm - 10 nm, 7 nm - 10 nm, 7.5 nm - 10 nm, 8 nm - 10 nm, 8.5 nm - 10 nm, 9 nm - 10 nm, 9.5 nm - 10 nm from any other adjacent adjuvant molecule.

[0030] In some aspects, the present disclosure provides a nucleic acid nanostructure comprising a plurality of adjuvant molecules and a plurality of antigens, wherein each adjuvant molecule among the plurality of adjuvant molecules is evenly spaced approximately 4.3 nm from any other adjuvant molecule.

[0031] In some embodiments, the nucleic acid nanostructure comprises at least 5, at least 10, at least 20, or at least 40 antigen - capturing motifs (e.g., coiled - coil peptides (CCP)). In some embodiments, the nucleic acid nanostructure comprises at least 5 antigen - capturing motifs. For example, the nucleic acid nanostructure may comprise 5 - 55, 5 - 50, 5 - 45, 5 - 40, 5 - 35, 5 - 30, 5 - 25, 5 - 20, 10 - 55, 10 - 50, 10 - 45, 10 - 40, 10 - 35, 10 - 30, 10 - 25, 10 - 20, 15 - 55, 15 - 50, 15 - 45, 15 - 40, 15 - 35, 15 - 30, 15 - 25, or 15 - 20 antigen - capturing motifs. In some embodiments, the nucleic acid nanostructure comprises 100 to 200, 150 to 200, 100 to 150, 50 to 100, 25 to 75, 25 to 50, 5 to 25, 10 to 25, or 15 to 25 antigen - capturing motifs. In some embodiments, the nucleic acid nanostructure comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 42, or 45 antigen - capturing motifs.

[0032] In some embodiments, each antigen - capturing motif among the plurality of antigen - capturing motifs (e.g., CCP) is evenly spaced 4.0 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, or 7 nm from any other adjacent antigen - capturing motif. In some embodiments, each antigen - capturing motif among the plurality of antigen - capturing motifs is spaced 8.6 - 10 nm, 4 nm - 50 nm, 5 nm - 50 nm, 4 nm - 40 nm, 4 nm - 30 nm, 4 nm - 25 nm, 4 nm - 20 nm, 4 nm - 15 nm, 5 nm - 15 nm, 4 nm - 10 nm, 4.5 nm - 10 nm, 5 nm - 10 nm, 5.5 nm - 10 nm, 6 nm - 10 nm, 6.5 nm - 10 nm, 7 nm - 10 nm, 7.5 nm - 10 nm, 8 nm - 10 nm, 8.5 nm - 10 nm, 9 nm - 10 nm, 9.5 nm - 10 nm from any other adjacent antigen - capturing motif.

[0033] In some aspects, the present disclosure provides a nucleic acid nanostructure comprising a plurality of antigen - capturing motifs (e.g., CCP) and a plurality of antigens, wherein each antigen - capturing motif among the plurality of antigen - capturing motifs is spaced 8.6 - 10 nm from any other antigen - capturing motif.

[0034] In some aspects, the present disclosure provides a nucleic acid nanostructure comprising a plurality of adjuvant molecules, a plurality of antigen capture motifs, and / or a plurality of antigens, wherein each adjuvant molecule of the plurality of adjuvant molecules and each antigen capture motif of the plurality of antigen capture motifs are evenly spaced by about 8.6 - 10 nm from any other adjuvant molecule and / or antigen capture motif.

[0035] In some embodiments, the distance between any two adjacent molecules of an adjuvant (e.g., CpG and / or dsRNA) is 2 nm - 10 nm. For example, the distance between any two adjacent molecules of an adjuvant (e.g., CpG and / or dsRNA) can be about 4 - 10 nm, 4 - 8 nm, or about 4 - 6 nm. In some embodiments, the distance between any two adjacent molecules of an adjuvant is 4 nm - 50 nm, 5 nm - 50 nm, 4 nm - 40 nm, 4 nm - 30 nm, 4 nm - 25 nm, 4 nm - 20 nm, 4 nm - 15 nm, 5 nm - 15 nm, 4 nm - 10 nm, 4.5 nm - 10 nm, 5 nm - 10 nm, 5.5 nm - 10 nm, 6 nm - 10 nm, 6.5 nm - 10 nm, 7 nm - 10 nm, 7.5 nm - 10 nm, 8 nm - 10 nm, 8.5 nm - 10 nm, 9 nm - 10 nm, 9.5 nm - 10 nm. In some embodiments, the distance between any two adjacent molecules of an adjuvant (e.g., CpG and / or dsRNA) is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 nm. The uniform spacing herein refers to the distance between any two adjacent molecules measured from the center of the molecule.

[0036] In some embodiments, the density of an adjuvant (e.g., CpG and / or dsRNA) on the nucleic acid nanostructure is 1 molecule of the adjuvant (e.g., CpG and / or dsRNA) per 5 to 50 nm 2 In some embodiments, the density of an adjuvant (e.g., CpG and / or dsRNA) on the nucleic acid nanostructure is 1 molecule of the adjuvant per 5 to 10 nm 2 1 molecule of the adjuvant per 5 to 20 nm 2 1 molecule of the adjuvant per 5 to 30 nm 2 1 molecule of the adjuvant per 5 to 40 nm 2 1 molecule of the adjuvant per 10 to 50 nm 2 1 molecule of the adjuvant per 20 to 50 nm 2 1 molecule of the adjuvant per 30 to 50 nm 2 1 molecule of the adjuvant per 40 to 50 nm 2 1 molecule of the adjuvant or per 20 to 40 nm 2There is 1 molecule of adjuvant. In some embodiments, the density of adjuvant (e.g., CpG and / or dsRNA) on the nucleic acid nanostructure is 1 molecule per 5 nm 2 There is 1 molecule, per 10 nm 2 There is 1 molecule, per 15 nm 2 There is 1 molecule, per 20 nm 2 There is 1 molecule, per 25 nm 2 There is 1 molecule, per 30 nm 2 There is 1 molecule, per 35 nm 2 There is 1 molecule, per 40 nm 2 There is 1 molecule, per 45 nm 2 There is 1 molecule or per 50 nm 2 There is 1 molecule.

[0037] In some embodiments, the distance between any two adjacent molecules of adjuvant (e.g., adjuvant molecule) (e.g., CpG and / or dsRNA) is about 4.3 nm. In some embodiments, the density of adjuvant (e.g., adjuvant molecule) (e.g., CpG and / or dsRNA) on the nucleic acid nanostructure is 1 molecule per 5 to 10 nm 2 There is 1 molecule of adjuvant (e.g., CpG and / or dsRNA). In some embodiments, the density of adjuvant (e.g., CpG and / or dsRNA) on the nucleic acid nanostructure is 1 molecule per 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 nm 2 There is 1 molecule of adjuvant (e.g., CpG and / or dsRNA).

[0038] In some embodiments, the distance between any two adjacent antigen capture motifs is 2 - 10 nm. For example, the distance between any two adjacent antigen capture motifs can be about 4 - 10 nm, 4 - 8 nm, or about 4 - 6 nm. In some embodiments, the distance between any two adjacent antigen capture motifs is 4 nm - 50 nm, 5 nm - 50 nm, 4 nm - 40 nm, 4 nm - 30 nm, 4 nm - 25 nm, 4 nm - 20 nm, 4 nm - 15 nm, 5 nm - 15 nm, 4 nm - 10 nm, 4.5 nm - 10 nm, 5 nm - 10 nm, 5.5 nm - 10 nm, 6 nm - 10 nm, 6.5 nm - 10 nm, 7 nm - 10 nm, 7.5 nm - 10 nm, 8 nm - 10 nm, 8.5 nm - 10 nm, 9 nm - 10 nm, 9.5 nm - 10 nm. In some embodiments, the distance between any two adjacent antigen capture motifs is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 nm. As used herein, uniform spacing refers to the distance between any two adjacent molecules measured from the center of the molecule or from the point of conjugation to the nanostructure.

[0039] In some embodiments, the density of antigen capture motifs on the nucleic acid nanostructure is 1 antigen capture motif per 5 to 50 nm 2 In some embodiments, the density of antigen capture motifs on the nucleic acid nanostructure is 1 antigen capture motif per 5 to 10 nm 2 1 antigen capture motif per 5 to 20 nm 2 1 antigen capture motif per 5 to 30 nm 2 1 antigen capture motif per 5 to 40 nm 2 1 antigen capture motif per 10 to 50 nm 2 1 antigen capture motif per 20 to 50 nm 2 1 antigen capture motif per 30 to 50 nm 2 1 antigen capture motif per 40 to 50 nm 2 1 antigen capture motif per 20 to 40 nm 2 In some embodiments, the density of antigen capture motifs on the nucleic acid nanostructure is 1 antigen capture motif per 5 nm 2 1 antigen capture motif per 10 nm 2 1 antigen capture motif per 15 nm 2 1 antigen capture motif per 20 nm 2 1 antigen capture motif per 25 nm 2 1 antigen capture motif per 30 nm 2 1 antigen capture motif per 35 nm2 There is 1 antigen - capturing motif per 40 nm 2 There is 1 antigen - capturing motif per 45 nm 2 There is 1 antigen - capturing motif or per 50 nm 2 There is 1 antigen - capturing motif.

[0040] In some embodiments, the distance between any two adjacent molecules of an adjuvant (e.g., CpG and / or dsRNA) and / or CCP is 2 nm - 10 nm. For example, the distance between any two adjacent molecules of an adjuvant (e.g., CpG and / or dsRNA) and / or CCP can be about 4 - 10 nm, 4 - 8 nm, or about 4 - 6 nm. In some embodiments, the distance between any two adjacent molecules of an adjuvant (e.g., CpG and / or dsRNA) and / or CCP is 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 nm. As used herein, uniform spacing refers to the distance between any two adjacent molecules measured from the center of the molecule.

[0041] In some embodiments, the density of an adjuvant (e.g., CpG and / or dsRNA) and / or CCP on the nucleic acid nanostructure is 1 molecule per 5 to 50 nm 2 There is 1 molecule of an adjuvant (e.g., CpG and / or dsRNA) or CCP. In some embodiments, the density of an adjuvant (e.g., CpG and / or dsRNA) and / or CCP on the nucleic acid nanostructure is 1 molecule per 5 nm 2 There is 1 molecule per 10 nm 2 There is 1 molecule per 15 nm 2 There is 1 molecule per 20 nm 2 There is 1 molecule per 25 nm 2 There is 1 molecule per 30 nm 2 There is 1 molecule per 35 nm 2 There is 1 molecule per 40 nm 2 There is 1 molecule per 45 nm 2 There is 1 molecule or per 50 nm 2 There is 1 molecule.

[0042] In some embodiments, the distance between any two adjacent molecules of an adjuvant (e.g., an adjuvant molecule) (e.g., CpG and / or dsRNA) and / or CCP is about 4.3 nm. In some embodiments, the density of an adjuvant (e.g., an adjuvant molecule) (e.g., CpG and / or dsRNA) and / or CCP on the nucleic acid nanostructure is 1 molecule per 5 to 10 nm 2There is 1 molecule of adjuvant (e.g., CpG and / or dsRNA) or CCP. In some embodiments, the density of adjuvant (e.g., CpG and / or dsRNA) and / or CCP on the nucleic acid nanostructure is per 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 nm 2 There is 1 molecule of adjuvant (e.g., CpG and / or dsRNA) or CCP.

[0043] In some embodiments, the nucleic acid nanostructure is a nucleic acid (e.g., DNA) origami nanostructure. In some embodiments, the nucleic acid nanostructure is a nucleic acid (e.g., DNA) single-stranded tile (SST) nanostructure.

[0044] In some embodiments, the plurality of antigens includes ovalbumin.

[0045] In some embodiments, the plurality of antigens are covalently linked to the nanostructure. In some embodiments, the plurality of antigens are covalently linked to the free amino groups of the nucleic acid nanostructure.

[0046] In some aspects, the present disclosure provides a method for inducing a Th1-type polarized immune response in a cell, the method comprising administering to a subject (e.g., a human subject) the nucleic acid nanostructure provided herein.

[0047] In some aspects, the present disclosure provides a method for inducing a Th1-type polarized immune response in a cell, the method comprising administering to a subject a nucleic acid nanostructure comprising a plurality of evenly spaced adjuvant molecules and a plurality of antigen molecules.

[0048] In some embodiments, the volume of the tumor is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold relative to a control (e.g., where the control is free CpG + free antigen + free nanostructure, or where the control is only buffer). In some embodiments, the volume of the tumor is reduced by 2-fold, 3-fold, 4-fold or 5-fold relative to the control.

[0049] In some embodiments, the nucleic acid nanostructure is administered to the subject multiple times (e.g., at least 2 times, at least 3 times, etc.).

[0050] Nucleic acid nanostructure

[0051] As used herein, "nucleic acid nanostructure" refers to a nucleic acid that forms (e.g., self-assembles into) a two-dimensional (2D) or three-dimensional (3D) shape (e.g., reviewed in W.M. Shih, C. Lin, Curr. Opin. Struct. Biol. 20, 276 (2010), which is incorporated herein by reference). Any nucleic acid folding or hybridization method can be used to form the nanostructure. One such method is DNA origami (see, e.g., Rothmund, P.W.K. Nature 440(7082):297-302 (2006), which is incorporated herein by reference). In the DNA origami method, a longer "scaffold" nucleic acid strand is folded by hybridizing it with multiple shorter "staple" oligonucleotides, each of which hybridizes to two or more non-consecutive regions within the scaffold strand, thereby generating a nanostructure. In some embodiments, the scaffold strand has a length of at least 100 nucleotides. In some embodiments, the scaffold strand has a length of at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, or at least 8000 nucleotides. The scaffold strand can be naturally occurring or non-naturally occurring. The staple strands typically have a length of less than 100 nucleotides; however, depending on the application and the length of the scaffold strand, they can be longer or shorter. In some embodiments, the staple strands can have a length of 15 to 100 nucleotides. In some embodiments, the staple strands have a length of 25 to 50 nucleotides.

[0052] In some embodiments, nucleic acid nanostructures are assembled in the absence of a scaffold strand (e.g., scaffold-free structures). For example, multiple oligonucleotides (e.g., having a length of less than 200 nucleotides or less than 100 nucleotides) can be assembled to form a nucleic acid nanostructure.

[0053] Other methods for assembling nucleic acid nanostructures are also known in the art, and any of them can be used herein. Such methods are described, for example, in Bellot G. et al., Nature Methods, 8:192-194 (2011); Liedl T. et al., Nature Nanotechnology, 5:520-524 (2010); Shih W.M. et al., Curr. Opin. Struct. Biol., 20:276-282 (2010); Ke Y. et al., J. Am. Chem. Soc, 131:15903-08 (2009); Dietz H. et al., Science, 325:725-30 (2009); Hogberg B. et al., J. Am. Chem. Soc, 131:9154-55 (2009); Douglas S.M. et al., Nature, 459:414-418 (2009); Jungmann R. et al., J. Am. Chem. Soc, 130:10062-63 (2008); Shih W.M., Nature Materials, 7:98-100 (2008); and Shih W.M., Nature, 411:618-21 (2004), the entire contents of each of which are incorporated herein by reference.

[0054] The nucleic acid nanostructures can be assembled into one of many defined and predetermined shapes, including but not limited to capsule-like, hemispherical, cubic, cuboid, tetrahedral, cylindrical, conical, octahedral, prismatic, spherical, pyramidal, dodecahedral, tubular, irregular, and abstract shapes. The nanostructures can have a void volume (e.g., it can be partially or completely hollow). In some embodiments, the void volume can be at least 25%, at least 50%, at least 75%, at least 85%, at least 90% or more of the nanostructure volume. Thus, in some embodiments, the nucleic acid nanostructures do not contain a solid core. In some embodiments, the shape of the nucleic acid nanostructures is not circular or approximately circular. In some embodiments, the nucleic acid nanostructures are not solid spheres. Depending on the intended use, the nucleic acid nanostructures can be assembled into shapes as simple as two-dimensional sheets or as complex as three-dimensional capsules or lattices (or even more complex).

[0055] In some embodiments, the nucleic acid nanostructures include barrel-shaped structures. See, for example Figures 2A - 2B。A description of the three-dimensional DNA barrel structure is described in Wickham et al., Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard. Nature Comm. (2020) 11:5768, the entire content of which is incorporated herein. In some embodiments, the diameter of the DNA barrel is from 30 to 120 nm. As used herein, the diameter of the DNA barrel is measured from the midpoint of the helix to the outer side of the nucleic acid (e.g., DNA). For example, the diameter of the DNA barrel can be 30 nm (see, e.g., Figure 4A), 60 nm, or 90 nm. In some embodiments, the diameter of the DNA barrel is from 30 nm to 120 nm, from 40 nm to 110 nm, from 50 nm to 100 nm, from 60 nm to 90 nm, or from 70 nm to 80 nm. In some embodiments, the diameter of the DNA barrel is 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 nm.

[0056] In some embodiments, the length of the DNA barrel is from 15 to 250 nm. As described herein, the height of the DNA barrel is measured along the Y-axis of the cylindrical nanostructure, as shown in Figure 4A. For example, the height of the DNA barrel can be from 15 nm to 250 nm, from 20 nm to 240 nm, from 30 nm to 230 nm, from 40 nm to 220 nm, from 50 nm to 210 nm, from 60 nm to 200 nm, from 70 nm to 190 nm, from 80 nm to 180 nm, from 90 nm to 170 nm, from 100 nm to 160 nm, from 110 nm to 150 nm, from 120 nm to 140 nm, or from 125 nm to 135 nm. In some embodiments, the height of the DNA barrel is 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 nm.

[0057] The barrel structure as described herein contains an outer (i.e., external) surface and an inner (i.e., internal) surface. As used herein, the outer surface of the DNA barrel refers to the portion of the nanostructure that faces the raised regions of the structure. The inner surface of the DNA barrel refers to the portion of the nanostructure that faces the concave regions of the structure (see, e.g., Figure 1, left side; Figure 4A, upper left; Figure 4C). A useful feature of the DNA barrel is the ability to control the physical availability of molecules attached to and / or incorporated within the nanostructure. For example, the CCP located on the inner surface of the DNA barrel can be shielded from interacting with other objects, while the adjuvants (e.g., CpG and / or dsRNA) and / or antigens located on the outer surface of the DNA barrel are exposed to the environment, allowing them to interact with other objects such as molecules, cells, nucleic acids, etc. In some embodiments, the CCP is located on the inner surface of the DNA barrel, and the adjuvants (e.g., CpG and / or dsRNA) and / or antigens are located on the outer surface of the DNA barrel.

[0058] Methods for assembling DNA barrel structures are known in the art, and any of them can be used herein. In some embodiments, the assembly of the DNA barrel includes mixing a scaffold strand with a ten-fold excess of staple strands in a folding buffer containing 5 mM Tris, 1 mM EDTA, and 6 - 20 mM MgCl2, followed by annealing the sample. In some embodiments, the sample can be annealed using the following annealing gradients: 65 - 25 °C for 18 to 72 hours, 65 °C for 15 minutes and 50 - 40 °C for 18 to 72 hours, or 65 °C for 15 minutes and 47 °C for 18 to 72 hours. For example, the folding buffer can contain 8 mM MgCl2, and the sample can be annealed at 47 °C for 18 hours. In other embodiments, the folding buffer can contain 10 mM MgCl2, and can be annealed at 65 °C for 15 minutes and then linearly decreased from 50 °C to 40 °C for 66 hours or 72 hours. In some embodiments, the folding buffer contains 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mM MgCl2.

[0059] In some embodiments, the DNA barrels are purified by methods known in the art. For example, the DNA barrels can be purified by rate-zonal centrifugation using a 15-45% (v / v) glycerol gradient, wherein the glycerol solution is prepared in TE buffer containing 10 mM MgCl2 and spun at 40,000 to 55,000 rpm for 25 minutes to 1 hour at 4°C. In some embodiments, the DNA barrels are purified by PEG precipitation. For example, the DNA barrels can be purified by mixing with a 10%-20% PEG solution (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% PEG) in a 1:1 ratio, wherein the PEG buffer is prepared in TE buffer containing salts (e.g., 12 mM MgCl2, 510 mM NaCl2), followed by centrifugation (e.g., spun at 16,000 rcf for 25 minutes at 22°C). In some embodiments, the DNA barrels are spun at 41,000 rpm for 25 minutes at 4°C. In other embodiments, the DNA barrels are spun at 55,000 rpm for 1 hour at 4°C. In some embodiments, the DNA barrels are spun at 40,000, 42,500, 45,000, 47,500, 50,000, 52,500, or 55,000 rpm. In some embodiments, the DNA barrels are spun for 25, 30, 35, 40, 45, 50, 55, or 60 minutes.

[0060] In some embodiments, the nucleic acid nanostructures include two-dimensional or three-dimensional square-lattice structures. An illustration of a three-dimensional square-lattice structure is described in Yonggang Ke et al., Multilayer DNA Origami Packed on a Square Lattice. J Am Chem Soc. November 4, 2009; 131(43):15903-8, the entire content of which is incorporated herein by reference.

[0061] The nucleic acid nanostructures can be made of, or comprise, DNA, RNA, modified DNA, modified RNA, PNA, LNA, or combinations thereof.

[0062] In some embodiments, nucleic acid nanostructures are rationally designed. A nucleic acid nanostructure is considered to be "rationally designed" herein if the nucleic acids forming the nanostructure are selected based on pre-determined, predictable nucleobase pairing interactions that direct nucleic acid hybridization. For example, a nucleic acid nanostructure can be designed prior to its synthesis, and certain selected nucleotides (e.g., oligonucleotides) can be used during synthesis to specify and control its size, shape, complexity, and modifications. The position of each nucleic acid within the structure can be known and provided prior to the synthesis of a nanostructure of a particular shape. For example, the basic principle of designing a self-assembling nucleic acid nanostructure is to select sequence complementarity in nucleic acid strands such that, by pairing complementary segments, the nucleic acid strands self-organize into a predefined nanostructure under appropriate physical conditions. Thus, in some embodiments, the nucleic acid nanostructures are self-assembling. Similarly, in some embodiments, the stem and anti-stem region nucleic acids (e.g., those linked to an adjuvant and / or antigen) can be rationally designed to specifically attach to the inner or outer surface of the nanostructure without intercalating or hybridizing with the nucleic acids forming the body of the nanostructure.

[0063] Examples of nucleic acid nanostructures used in accordance with the present disclosure include, but are not limited to, capsules, lattices (E. Winfree et al., Nature 394, 539 (1998); H. Yan et al., Science 301, 1882 (2003); H. Yan et al., Proc. Natl. Acad. of Sci. USA 100, 8103 (2003); D. Liu et al., J. Am. Chem. Soc. 126, 2324 (2004); P. W. K. Rothemund et al., PLoS Biology 2, 2041 (2004)), ribbons (S. H. Park et al., NanoLett. 5, 729 (2005); P. Yin et al., Science 321, 824 (2008)), tubes (H. Yan, Science (2003); P. Yin (2008)), finite two-dimensional (2D) and three-dimensional (3D) objects with defined shapes (J. Chen, N. C. Seeman, Nature 350, 631 (1991); P. W. K. Rothemund, Nature 440, 297 (2006); Y. He et al., Nature 452, 198 (2008); Y. Ke et al., Nano. Lett. 9, 2445 (2009); S. M. Douglas et al., Nature 459, 414 (2009); H. Dietz et al., Science 325, 725 (2009); E. S. Andersen et al., Nature 459, 73 (2009); T. Liedl et al., Nature Nanotech. 5, 520 (2010); D. Han et al., Science 332, 342 (2011)), and macroscopic crystals (J. P. Meng et al., Nature 461, 74 (2009)). Other nucleic acid nanostructures as provided herein can also be used.

[0064] Cadnano software can be used to design specific nucleic acid nanostructures of interest (see cadnano.org).

[0065] As used herein, the terms "nucleic acid" and / or "oligonucleotide" can refer to at least two nucleotides covalently linked together. The nucleic acids of the present disclosure typically can contain phosphodiester bonds, but in some cases, include nucleic acid analogues that can have other backbones, including, for example, phosphoramidates (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioates (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Patent No. 5,644,048), phosphorodithioates (Brill et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated herein by reference).Other similar nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)); nonionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ACS Symposium Series 58, “Carbohydrate Modifications in Antisense Research,” edited by Y.S. Sanghui and P.D. Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and those with non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506 and Chapters 6 and 7, ACS Symposium Series 58, “Carbohydrate Modifications in Antisense Research,” edited by Y.S. Sanghui and P.D. Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp. 169-176). Several nucleic acid analogs are described in Rawls, C&E News, June 2, 1997, p. 35. All of these references are hereby expressly incorporated herein by reference. Nucleic acids can have homogeneous backbones (e.g., entirely phosphodiester or entirely phosphorothioate) or heterogeneous (or chimeric) backbones. Under certain conditions, phosphorothioate backbone modifications render nucleic acids more resistant to nucleases and thus more stable (compared to native phosphodiester backbone nucleic acids). Other linkages that can provide more stability to nucleic acids include, but are not limited to, dithiophosphonate linkages, methylphosphonate linkages, methylthiophosphonate linkages, boranophosphate linkages, peptide linkages, alkyl linkages, dephospho linkages, etc. Thus, in some cases, nucleic acids have non-naturally occurring backbones.The phospho-ribose backbone can be modified, e.g., to facilitate the addition of labels or to increase the stability and half-life of such molecules in physiological environments.

[0066] The nucleic acid can be single-stranded (ss) or double-stranded (ds), as specified, or can contain portions of both single-stranded and double-stranded sequences (e.g., be partially double-stranded). The nucleic acid can be DNA (both genomic and cDNA), RNA, or a hybrid, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. As used herein, the term "nucleoside" includes nucleotides, as well as nucleoside and nucleotide analogs, and modified nucleosides, such as amino-modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus, for example, the individual units of peptide nucleic acid, each containing a base, are referred to herein as nucleosides.

[0067] Nucleic acids include DNA such as B-form DNA, D-form DNA, and L-form DNA, and RNA, as well as their various modifications. Modifications include base modifications, sugar modifications, and backbone modifications. Non-limiting examples of such nucleic acids are provided below.

[0068] Non-limiting examples of DNA variants that can be used as provided herein are L-DNA (a DNA backbone enantiomer known in the literature), peptide nucleic acid (PNA) bisPNA clamp structures, pseudocomplementary PNA, locked nucleic acid (LNA), or co-nucleic acids of the foregoing, such as DNA-LNA co-nucleic acids. It should be understood that the nucleic acids used as provided herein can be homogeneous or heterogeneous in nature. For example, they may be entirely DNA in nature, or they may contain DNA and non-DNA (e.g., LNA) monomers or sequences. Thus, any combination of nucleic acid elements can be used. Nucleic acid modifications can render the nucleic acid more stable and / or less degradable under certain conditions. For example, in some cases, the nucleic acid is nuclease-resistant.

[0069] Methods for synthesizing nucleic acids (e.g., ssDNA or dsDNA, or ssRNA or dsRNA) are known in the art and are described, for example, in U.S. Patent Nos. 5,143,854 and 5,445,934, the entire contents of which are incorporated herein.

[0070] Nucleic acids can be synthesized in vitro. Methods for synthesizing nucleic acids, including automated nucleic acid synthesis, are also known in the art. Nucleic acids having modified backbones, such as those containing phosphorothioate linkages, and including those containing chimeric modified backbones, can be synthesized using automated techniques employing phosphoramidite or H-phosphonate chemistries. (F.E. Eckstei, "Oligonucleotides and Analogu–s-APractical Appro”ch", IRL Press, Oxford, UK, 1991 and M.D. Matteucci and M.H. Caruthers, Tetrahedron Lett. 21, 719 (1980)). Aryland alkylphosphonate linkages can be prepared, for example, as described in U.S. Patent No. 4,469,863; and alkyl phosphotriester linkages (wherein the charged oxygen moiety is alkylated), for example, as described in U.S. Patent No. 5,023,243 and European Patent No. 092,574, can be prepared by automated solid-phase synthesis using commercially available reagents. Methods for effecting other DNA backbone modifications and substitutions have been described. Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J (1990) Bioconjugate Chem 1:165; Crooke ST et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; and Hunziker J et al. (1995) Mod Synth Methods 7:331-417.

[0071] Nucleic acids can additionally or alternatively contain modifications in their sugars. For example, a β-ribose unit or a β-’-2’-deoxyribose unit can be replaced with a modified sugar unit, where the modified sugar unit is, for example, selected from β-D-ribose, α-’-2’-deoxyribose, ’-2’-deoxyribose, 2’-’2’-deoxyribose, arabinose, 2’-F-arabinose, 2’-O-(C1-C6)alkyl ribose, preferably, 2’-O-(C1-C6)alkyl ribose is 2’-O-methyl ribose, 2’-O-(C2-C6)alkenyl ribose, 2’-[O-(C1-C6)alkyl-O-(C1-C6)alkyl]-ribose, 2’-NH ’-2'-deoxyribose, β-D-xylo-furanose, α-L-arabinofuranose, 2,4-dideoxy-β-D-erythro-hexopyranose and carbocyclic sugars (e.g., described in Froehler J (1992) Am Chem Soc 114:8320) and / or acyclic sugar analogs (e.g., described in Vandendriessche et al. (1993) Tetrahedron 49:7223) and / or bicyclic sugar analogs (e.g., described in Tarkov M et al. (1993) Helv Chim Acta 76:481).

[0072] Nucleic acids can contain modifications in their bases. Modified bases include modified cytosines (such as 5-substituted cytosines (e.g., 5-methylcytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-difluoromethylcytosine, and unsubstituted or substituted 5-alkynylcytosine), 6-substituted cytosines, N4-substituted cytosines (e.g., N4-ethylcytosine), 5-azacytosine, 2-mercaptocytosine, isocytosine, pseudoisocytosine, cytosine analogs with fused ring systems (e.g., N,N’-propylenecytosine or phenoxazine), and uracil and its derivatives (e.g., 5-fluorouracil, 5-bromouracil, 5-bromovinyluracil, 4-thiouracil, 5-hydroxyuracil, 5-propynyluracil), modified guanines, such as 7-deazaguanine, 7-deaza-7-substituted guanines (such as 7-deaza-7-(C2-C6) alkynylguanine), 7-deaza-8-substituted guanines, hypoxanthine, N2-substituted guanines (e.g., N2-methylguanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenines (e.g., N6-methyladenine, 8-oxoadenine), 8-substituted guanines (e.g., 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. Nucleic acids can contain universal bases (e.g., 3-nitropyrrole, P-base, 4-methylindole, 5-nitroindole, and K-base) and / or aromatic ring systems (e.g., fluorobenzene, difluorobenzene, benzimidazole, or dichloro-benzimidazole, 1-methyl-1H-[1,2,4]triazole-3-carboxamide). Specific base pairs that can be incorporated into the oligonucleotides of the present invention are the dZ and dP non-standard nucleobase pairs reported by Yang et al., NAR, 2006, 34(21):6095-6101. The pyrimidine analog dZ is 6-amino-5-nitro-3-(1’-β-D-2’-deoxyribofuranosyl)-2(1H)-pyridone, and its Watson-Crick complement dP, a purine analog, is 2-amino-8-(1’-β-D-1’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one.

[0073] In an exemplary embodiment, the nucleic acid nanostructure comprises single-stranded genomic DNA. For example, the nucleic acid nanostructure can comprise linear or circular single-stranded M13 plasmid DNA. In some embodiments, the nucleic acid nanostructure does not comprise plasmid DNA.

[0074] It should be understood that in some embodiments, the nucleic acid nanostructures of the present disclosure do not include aggregated nucleic acids. As used herein, "aggregated nucleic acids" refers to compact nucleic acids, e.g., nucleic acids that twist and coil upon themselves (see, e.g., Teif VB et al. Progress in Biophysics and Molecular Biology 105(3):208–222, incorporated herein by reference). The term "aggregated nucleic acids" does not include nucleic acid nanostructures having a unique 2D or 3D architecture.

[0075] It should also be understood that in some embodiments, the nucleic acid nanostructures of the present disclosure do not include coding nucleic acids. That is, in some embodiments, the nucleic acid nanostructures comprise non-coding nucleic acids (e.g., nucleic acids that do not encode a protein). As used herein, "coding nucleic acid" refers to a nucleic acid containing a nucleotide sequence that specifies the amino acid sequence of a given protein (e.g., a therapeutic protein). Thus, a "non-coding nucleic acid" is a nucleic acid that does not specify the amino acid sequence of a protein and thus does not transcribe into RNA or translate into a protein. In other embodiments, it should be understood that the nucleic acid nanostructures can comprise one or more coding nucleic acids.

[0076] In some embodiments, the nucleic acids used to prepare the nucleic acid nanostructures do not encode any amino acids. In some embodiments, the nucleic acids used to prepare the nucleic acid nanostructures do not encode more than 1, 2, 3, 4, or 5 consecutive amino acids.

[0077] In some embodiments, the nucleic acids used to prepare the nucleic acid nanostructures do not include regulatory elements / sequences well known in the art, such as promoters, enhancers, polyA sequences, and / or ribosome binding site sequences.

[0078] In some embodiments, the nucleic acids used to prepare the nucleic acid nanostructures are not plasmids.

[0079] In some embodiments, the nucleic acids used to prepare the nucleic acid nanostructures contain more than one nucleic acid, and the nucleic acids are different from each other. That is, the nucleic acids of the nucleic acid nanostructures can comprise multiple different nucleic acids.

[0080] In some embodiments, the nucleic acid nanostructures are not lipid-encapsulated or lipid-coated (e.g., not linked to lipids). For example, many gene delivery methods in the prior art utilize nucleic acid nanostructures that are linked to hydrophobic moieties and / or covered with lipids (e.g., such as lipid bilayers), which function to prevent nuclease degradation (see, e.g., WO2013148186 A1). In some embodiments, the present disclosure does not include nucleic acid nanostructures that are linked to hydrophobic moieties and / or covered with lipids. However, in other embodiments, the nucleic acid nanostructures may contain one or more nucleic acids linked to one or more hydrophobic moieties and / or lipids.

[0081] The nucleic acid nanostructures of the present disclosure have a variety of in vitro and in vivo uses. In some embodiments, the nucleic acid nanostructures are used as scaffolds, cages, or multifunctional carriers for delivering antigens intended for use in vivo and / or in vitro. The nucleic acid nanostructures can be delivered by any suitable delivery method, such as intravenous or oral delivery.

[0082] The present disclosure encompasses conferring addressability to the nucleic acid nanostructures. For example, the nucleic acid nanostructures can be modified by site-specific attachment of targeting moieties such as proteins, ligands, or other small biomolecules. In some embodiments, the nucleic acid nanostructures may contain nucleic acid "staple" strands as described above, which serve as handle regions to enable nanoscale-specific placement of auxiliary molecules (e.g., biotin / streptavidin) at almost any position on or within the structure (see, e.g., Stein et al. Chemphyschem. 12(3), 689–695 (2011); Steinhauer et al. Angew Chem. Int. Ed. Engl. 48(47), 8870–8873 (2009); Stein et al. J. Am. Chem. Soc. 133(12), 4193–4195 (2011); Kuzyk et al. Nature 483(7389), 311–314 (2012); and Ding et al. J. Am. Chem. Soc. 132(10), 3248–3249 (2010); Yan et al. Science 301(5641), 1882–1884 (2003); and Kuzuya et al. Chembiochem. 10(11), 1811–1815 (2009), each of which is incorporated herein by reference).

[0083] In some embodiments, the nanostructured nucleic acids provided herein are modified (e.g., covalently modified) with a linker (e.g., a biotin linker) during synthesis or by enzymatic means (see, e.g., Jahn et al. Bioconjug. Chem. 22(4), 819–823 (2011), incorporated herein by reference). Such methods can also be used to localize reaction systems on nucleic acid nanostructures through the chemical biotinylation of enzyme molecules (see, e.g., Voigt et al. Nat. Nanotechnol. 5(3), 200–203 (2010)).

[0084] More conventional antibody-based binding methods can also be used to attach target proteins to nucleic acid nanostructures at a defined distance (see, e.g., Williams et al. Angew Chem. Int. Ed. Engl. 46(17), 3051–3054 (2007); and He Y et al. J. Am. Chem. Soc. 128(39), 12664–12665 (2006), each incorporated herein by reference). Thus, in some embodiments, nucleic acid nanostructures are linked to one or more antibodies.

[0085] In other embodiments, DNA aptamers adopt specific secondary structures with high binding affinities for specific molecular targets and are used as linkers, thereby eliminating the need for protein linkers (see, e.g., Ellington et al. Nature 346(6287), 818–822 (1990); Chhabra et al. J. Am. Chem. Soc. 129(34), 10304–10305 (2007); and Rinker et al. Nat. Nanotechnol. 3(7), 418–422 (2008), each incorporated herein by reference).

[0086] The present disclosure also encompasses the use of recombinant genetic engineering methods to selectively add affinity tags or other peptide linkers to nucleic acid nanostructures. For example, a polyhistidine sequence consisting of multiple histidine residues at the C- or N-terminus of a target protein is a commonly used tag for affinity-based purification. This can in turn be linked to nitrilotriacetic acid molecules through nickel-mediated interactions, and the nitrilotriacetic acid molecules are covalently conjugated to amines (see, e.g., Goodman et al. Chembiochem. 10(9), 1551–1557 (2009), incorporated herein by reference) or thiol-modified (see, e.g., Shen et al. J. Am. Chem. Soc. 131(19), 6660–6661 (2009), incorporated herein by reference) nucleic acids. By this method, fluorescent proteins can be periodically and specifically positioned on nucleic acid nanostructures (Goodman et al. (2009); and Shen et al. (2009)). Similarly, SNAP and peptide sequences are also used for the affinity purification of recombinant proteins and can be used to decorate nucleic acid nanostructures with different protein or enzyme species (see, e.g., Sacca et al. Angew Chem. Int. Ed. Engl. 49(49), 9378–9383 (2010), which is incorporated herein by reference). A related method involves generating chimeric proteins conjugated to DNA-binding domains, which can obviate the usually complex chemical synthesis techniques and toxic compounds (e.g., nickel) necessary to stably conjugate affinity tag binding partners to oligonucleotide strands. Additionally, zinc finger domains that recognize specific double-stranded sequences can be used to align fluorescent proteins at specific positions on the nucleic acid nanostructures of the present disclosure (see, e.g., Nakata et al. Angew Chem. Int. Ed. Engl. 51(10), 2421–2424 (2012), which is incorporated herein by reference).

[0087] Adjuvants (e.g., CpG and / or dsRNA) and / or antigens can be covalently or non-covalently attached to nucleic acid nanostructures. The location and nature of the linkage between the adjuvant (e.g., CpG and / or dsRNA) and / or antigen and the nucleic acid nanostructure will depend on the function of the adjuvant (e.g., CpG and / or dsRNA) and / or antigen. For example, the adjuvant (e.g., CpG and / or dsRNA) and / or antigen may be designed to be released (including slow release) from the nanostructure, and in such cases, the linkage between the adjuvant (e.g., CpG and / or dsRNA) and / or antigen and the nanostructure can be selected to achieve the desired release profile. In some embodiments, the adjuvant (e.g., CpG and / or dsRNA) and / or antigen is inactive in its bound form and is only activated upon release.

[0088] In some embodiments, an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen is combined with a nucleic acid during the assembly (e.g., self-assembly) of the nanostructure, or an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen is combined with a preformed nucleic acid nanostructure.

[0089] An adjuvant (e.g., CpG and / or dsRNA) and / or an antigen can be attached to the inner surface (in an inner compartment) or the outer surface of the nanostructure. The adjuvant (e.g., CpG and / or dsRNA) and / or an antigen can be arranged in various configurations. In some embodiments, the adjuvant (e.g., CpG and / or dsRNA) and / or an antigen is indirectly attached to the nucleic acid nanostructure via a stem region / anti-stem region hybridization system. As used herein, the term "stem region" refers to an extension of a staple strand in a nucleic acid nanostructure. As used herein, the term "anti-stem region" refers to a nucleic acid sequence complementary to the nucleic acid stem region (i.e., the stem region and the anti-stem region can bind to each other). In some embodiments, the stem region and / or the anti-stem region is a double-stranded DNA molecule, a single-stranded DNA molecule, a single-stranded RNA molecule, or a double-stranded RNA molecule. In some embodiments, the stem region and / or the anti-stem region contains modified nucleotides (e.g., pseudouridine-5'-triphosphate, 5-methoxyuridine-5'-triphosphate, N1-methylpseudouridine-5'-triphosphate, etc.). The lengths of the stem region and the anti-stem region can vary. By way of example, the lengths of the stem region and the anti-stem region can be from 5 to 50 nucleotides. In some embodiments, the lengths of the stem region and the anti-stem region are 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In some embodiments, the anti-stem region can be chemically conjugated to the adjuvant (i.e., CpG and / or dsRNA) and / or the antigen such that when the stem region hybridizes (i.e., complementary binds) to the anti-stem region, the adjuvant (e.g., CpG and / or dsRNA) and / or the antigen becomes indirectly linked to the nucleic acid nanostructure. It should be understood that the nanostructures of the present disclosure allow for the precise placement of one adjuvant (e.g., CpG and / or dsRNA) and / or an antigen or more than one adjuvant (e.g., CpG and / or dsRNA) and / or an antigen (e.g., a combination of different adjuvants (e.g., CpG and / or dsRNA) and / or antigens) on the inner surface and / or the outer surface of the nanostructure.

[0090] The nucleic acid nanostructures of the present disclosure allow for the high-density "packaging" of an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen on and in the nanostructure. In some embodiments, the nucleic acid nanostructure is decorated with one adjuvant (e.g., CpG or dsRNA) and / or an antigen per 50 nm 2 to 75 nm 2 In some embodiments, the nucleic acid nanostructure is decorated with one adjuvant (e.g., CpG or dsRNA) and / or an antigen per 50 nm 2 、55 nm 2 、60 nm2 , 65 nm 2 , 70 nm 2 or 75 nm 2 decorated with an adjuvant (e.g., CpG or dsRNA) and / or an antigen. For example, for a cylindrical nanostructure that is 30 nm tall and 60 nm in diameter, using a rhombic lattice spacing, 72 positions on the outside and 84 positions on the inside of the nanostructure can be occupied by an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen. For larger nanostructures, e.g., those having two 30 nm × 60 nm cylindrical nanostructures, the number of positions occupied by an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen is doubled. For even larger nanostructures, e.g., those having three 30 nm × 60 nm cylindrical nanostructures, the number of positions occupied by an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen is tripled, and so on.

[0091] In some aspects, the present disclosure encompasses systemic delivery or delivery to a local region, tissue, or cell of a nucleic acid nanostructure or a nucleic acid nanostructure loaded with an adjuvant (e.g., CpG and / or dsRNA) and / or an antigen. Any adjuvant (e.g., CpG and / or dsRNA) and / or antigen agent can be delivered using the methods of the present disclosure provided that it can be loaded onto or into the nucleic acid nanostructure. Since such processes are relatively harmless, it is expected that almost any adjuvant (e.g., CpG and / or dsRNA) and / or antigen can be used.

[0092] The length of the CpG adjuvant can vary. For example, the length can be 10 - 100 nucleotides (nt), 10 - 50 nt, or 10 - 20 nt.

[0093] adjuvant

[0094] "Adjuvant" is an agent that enhances the immune response against an antigen. In some embodiments, the adjuvant is a CpG oligonucleotide. A CpG oligonucleotide is a short single-stranded synthetic DNA molecule that contains deoxycytidine triphosphate ("C") followed by deoxyguanosine triphosphate ("G"). "p" refers to a phosphodiester or modified phosphorothioate (PS) bond linkage between consecutive nucleotides. CpG oligonucleotides generally enhance the immunostimulatory effect of nucleic acid nanostructures (Li, J. et al. ACS NANO, 5(11):8783 - 8789, 2011; Schuller, V. et al. ACS NANO, 5(12):9696 - 9702, 2011). For example, CpG oligonucleotides are signatures of microbial DNA and, after their uptake by cells, are recognized by endosomal Toll-like receptor 9 (TLR9), thereby activating downstream pathways to induce an immunostimulatory effect and resulting in a high-level secretion of various pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12. In some embodiments, the CpG oligonucleotide is linked to the internal surface of the nucleic acid nanostructure. In some embodiments, the CpG oligonucleotide is linked to the external surface of the nucleic acid nanostructure. In some embodiments, the nucleic acid nanostructure has CpG oligonucleotides linked to both the internal and external surfaces. Other examples of adjuvants include, but are not limited to, lipopolysaccharide and polyI:C (dsRNA mimetic).

[0095] Antigen

[0096] "Antigen" as used herein is any biomolecule that induces an immune response. In some embodiments, the antigen is a peptide, protein, or polypeptide, or a nucleic acid. In some embodiments, the antigen comprises a mixture of proteins derived from tumor cells. In some embodiments, the antigen is a cancer antigen. A cancer antigen is a type of protein or molecule produced by cancer cells and detectable by the immune system. A cancer antigen can be, for example, a component or element of a cancer cell or a biomolecule isolated from a cancer cell (e.g., a biomolecule known to be associated with a cancerous tumor). In some embodiments, the cancer antigen comprises a biomolecule (e.g., a peptide or polypeptide) that is overexpressed or overactivated in cancer cells relative to normal and non-cancerous cells. In some embodiments, the antigen is associated with an infectious disease. In some embodiments, the antigen associated with an infectious disease is a type of protein or molecule produced by a viral particle or cell associated with the infectious disease.

[0097] Non-limiting examples of cancer antigens include Her2 peptides (for vaccination against selected breast cancers); NY-ESO-1 peptides (for vaccination against selected bladder cancers); HPV16 E7 peptides (for vaccination against selected cervical cancers); carcinoembryonic antigen (for vaccination against selected colorectal cancers); Wilms tumor 1 (WT1) peptides (for vaccination against selected leukemias); MART-1, gp100, and tyrosinase (for vaccination against selected melanomas); URLC10, VEGFR1, and VEGFR2 (for vaccination against selected non-small cell lung cancers); survivin (for vaccination against selected ovarian cancers); MUC1 (for vaccination against selected pancreatic cancers); MUC2 (for vaccination against selected prostate cancers); telomerase (TERT); indoleamine 2,3-dioxygenase (IDO1); CTAG1B, and VEGF receptors (FLT1 and KDR). In some embodiments, the cancer antigens are as described in the following documents, namely Tagliamonte, M. et al. “Antigen-specific vaccines for cancer treatment”, Hum Vaccin Immunother. November 2014; 10(11):3332–3346.; or Pol, J. et al. “Trial Watch: Peptide-based anticancer vaccines”, Oncoimmunology. April 2015; 4(4):e974411.

[0098] In some embodiments, the cancer antigen is selected from the following antigens: CEA; gp100; Pmell7; mammaglobin-A; Melan-A; MART-1; NY-BR-1; ERBB2; OA1; PAP; PSA; RAB38; NY-MEL-1; TRP-1; gp75; TRP-2; tyrosinase; WT1; CD33; BAGE-1; D393-CD20n; cyclin-A1; GAGE-1,2,8; GAGE-3,4,5,6,7; GnTVf; HERV-K-MEL; KK-LC-1; KM-HN-1; LAGE-1; LY6K; MAGE-A1; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-A10; MAGE-A12m; MAGE-C1; MAGE-C2; mucink; NA88-A; NY-ESO-1; LAGE-2; SAGE; Spl7; SSX-2; SSX-4; survivin; BIRC5; TAG-1; TAG-2; TRAG-3; TRP2-INT2g; XAGE-lb; GAGED2a; BCR-ABL(b3a2); adipophilin; AIM-2; ALDH1A1; BCLX(L); BING-4; CALCA; CD45; CD274; CPSF; cyclin D1; DKKl; ENAH(hMena); EpCAM; EphA3; EZH2; FGF5; glypican-3; G250; MN; CAIX; HER-2; neu; HLA-DOB; hepsin; IDUA; IGF2B3; IL13Rα2; intestinal carboxylesterase; alpha-foetoprotein; kallikrein 4; KIF20A; Lengsin; M-CSF; MCSP; mdm-2; Meloe; Midkine; MMP-2; MMP-7; MUC1; MUC5AC; p53; PAX5; PBF; PRAME; PSMA; RAGE-1; RGS5; RhoC; RNF43; RU2AS; secernin 1; SOX10; STEAP1; telomerase; TPBG; and VEGF.

[0099] Antigen capture motif

[0100] As used herein, "antigen-capturing motif" refers to any biomolecule that binds an antigen (e.g., neoantigen). In some embodiments, the antigen-capturing motif is a nucleic acid (e.g., DNA and / or RNA), a peptide, and / or a protein. In some embodiments, the antigen-capturing motif is hydrophobic. In some embodiments, the hydrophobic antigen-capturing motif is capable of binding and capturing hydrophobic immunogenic neoantigens. In some embodiments, the antigen-capturing motif is hydrophilic. In some embodiments, the hydrophilic antigen-capturing motif is capable of binding and capturing hydrophilic immunogenic neoantigens. In some embodiments, the antigen-capturing motif is uncharged, negatively charged, or positively charged.

[0101] In some embodiments, the antigen-capturing motif is attached to the DNA nanostructure through its N-terminus. In some embodiments, the antigen-capturing motif is attached to the DNA nanostructure through its C-terminus.

[0102] In some embodiments, the antigen-capturing motif is a coiled-coil peptide (see, e.g., FIG. 6A). A coiled-coil peptide (CCP) is a common structural motif consisting of two or more α-helices that wind around each other to form a superhelical bundle. There can be two, three, or four helices in the bundle, and they can be arranged in the same (parallel) or opposite (antiparallel) directions. Examples of proteins containing the coiled-coil motif include, but are not limited to, myosin, tropomyosin, intermediate filaments, keratin, fibrinogen, c-Fos, and c-Jun. The amino acid sequence of a peptide that forms a coiled-coil bundle is characterized by a heptapeptide repeat unit, denoted as (abcdefg) n where n is the number of repeats. The interaction between the helices reduces the typical α-helix pitch from 3.6 residues per turn to 3.5 residues, creating an interfacial stripe between the associated helices where the residues in the a and d positions are typically hydrophobic. This forms the core of the coiled-coil through the packing of hydrophobic amino acids, and the core is stabilized by hydrophobic and van der Waals interactions. Importantly, the type of hydrophobic amino acids present in the interface can specify the number of helices in each superhelical bundle. The e and g positions tend to be occupied by polar / charged amino acids, which together form complementary charge pairs in the bundle, and these charge pairs contribute to the stabilization of the coiled-coil through interchain electrostatic interactions. Both the hydrophobic interactions caused by the residues in the a and d positions and the electrostatic interactions between the residues in the e and g positions can be used to influence the oligomeric state, parallel versus antiparallel topology, registration, and thermodynamic stability of the bundle. The residues in the b, c, and f positions can be used to provide sufficient solubility to the peptide and to control the higher-order aggregation of the oligomer through the outer surface of the coiled-coil.

[0103] In some embodiments, the antigen-capturing motif (e.g., CCP) is 10 - 50 amino acids in length. In some embodiments, the antigen-capturing motif (e.g., CCP) is 15 to 45, 20 to 40, 25 to 35, or about 30 amino acids in length. In some embodiments, the antigen-capturing motif (e.g., CCP) is 10, 15, 20, 25, 30, 35, 40, 45, and / or 50 amino acids in length.

[0104] In some embodiments, the antigen-capturing motif (e.g., CCP) comprises a modified amino acid (e.g., ε-azidolysine (azK), 4-hydroxyproline, 5-hydroxylysine, 6-N-methyllysine, γ-carboxyglutamic acid, desmosine, selenocysteine, phosphoserine (pSer), phosphothreonine (pThr), phosphotyrosine (pTyr), sulfotyrosine, symmetric Arg(Me)2, asymmetric Arg(Me)2, Arg(Me), argpyrimidine, Asn(GlcNAc), MeLys, (Me)2Lys, (Me)3Lys, acLys, carboxymethyllysine, or Thr(GalNAc).

[0105] In some embodiments, the CCP attached to the DNA nanostructures provided herein has the following amino acid sequence:

[0106]

[0107]

[0108] * “Ac” represents acetyl; “NH2” represents amine

[0109] Method of Use

[0110] The nucleic acid nanostructure vaccines of the present disclosure can be used to provide therapeutic benefits (e.g., treatment) when administered to a subject. The nucleic acid nanostructure vaccines (e.g., nucleic acid barrel nanostructures conjugated with adjuvant molecules and antigen capture motifs) can be used to treat a disease in a subject (e.g., a human subject). In some embodiments, the nucleic acid nanostructure vaccines are used to treat cancer in a subject (e.g., a human subject). Thus, for example, in some embodiments, provided herein is a method of treating a subject having cancer by administering the nucleic acid nanostructure vaccines of the present disclosure. In some embodiments, the nucleic acid nanostructure vaccines are administered prophylactically to a subject (e.g., treating cancer by preventing tumor formation or progression). In some embodiments, provided herein is a method of treating a subject having an infectious disease by administering the nucleic acid nanostructure vaccines of the present disclosure. In some embodiments, the nucleic acid nanostructure vaccines are formulated into a pharmaceutical composition. In other embodiments, provided herein is a method of administering the nucleic acid nanostructure vaccines in an effective amount to generate a T cell immune response in a subject.

[0111] As used herein, the terms “treatment,” “treating,” and “therapy” refer to therapeutic treatment and prophylactic or preventive procedures. These terms also include ameliorating existing symptoms, preventing additional symptoms, alleviating or preventing the underlying cause of symptoms, preventing or reversing the cause of symptoms (e.g., symptoms associated with cancer or an infectious disease). Additionally, the term “treatment” also includes applying or administering a nucleic acid nanostructure vaccine to a subject having a disease or to an isolated tissue or cell line from a subject, with the aim of curing, restoring, alleviating, mitigating, altering, remedying, relieving, improving, or affecting the disease, disease symptoms, or disease predisposition.

[0112] In some embodiments, the nucleic acid nanostructure vaccine is administered prophylactically to a subject (e.g., before the onset of a disease or before the subject experiences symptoms of a disease such as cancer or an infectious disease). In some embodiments, the nucleic acid nanostructure vaccine is administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks or more before the onset of the disease. In some embodiments, the nucleic acid nanostructure vaccine is administered to the subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks or more before the subject experiences symptoms of the disease. In some embodiments, a prophylactic dose of the nucleic acid nanostructure vaccine provides a therapeutic benefit for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks or more after administration of the vaccine. In some embodiments, a prophylactic dose of the nucleic acid nanostructure vaccine provides a therapeutic benefit (e.g., preventing the onset of a disease such as cancer) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks or more after administration of the vaccine. In some embodiments, a prophylactic dose of the nucleic acid nanostructure vaccine provides a therapeutic benefit (e.g., preventing the onset of a disease such as cancer) for 1, 2, 3, 4, 5, 6, 7, 8, 9 months or more after administration of the vaccine. In some embodiments, a prophylactic dose of the nucleic acid nanostructure vaccine provides a therapeutic benefit (e.g., preventing the onset of a disease such as cancer) for 0-10 weeks, 1-10 weeks, 2-7 weeks, 2-5 weeks, 5-10 weeks or 5-20 weeks after administration of the vaccine. In some embodiments, a prophylactic dose of the nucleic acid nanostructure vaccine provides a therapeutic benefit (e.g., preventing the onset of a disease such as cancer) for at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150 or 200 days after administration of the vaccine.

[0113] Vaccination using the nucleic acid nanostructure vaccine of the present disclosure may include administration of a single dose, or administration of two or more doses. Thus, in some embodiments, vaccination includes administration of a first dose and administration of a second (booster) dose. An additional (booster) dose may be administered 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months or 1 year (e.g., annually) after the previous (e.g., first) dose.

[0114] In some embodiments, a nucleic acid nanostructure vaccine is administered to a subject having a disease (e.g., cancer or an infectious disease). In some embodiments, a nucleic acid nanostructure vaccine is administered to a former cancer patient who has no remaining signs of disease (e.g., no observable tumors). In some embodiments, administering a nucleic acid nanostructure vaccine to a former cancer patient provides a therapeutic benefit (e.g., no tumor recurrence) to the patient for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks or more after administration of the vaccine. In some embodiments, administering a nucleic acid nanostructure vaccine to a former cancer patient provides a therapeutic benefit (e.g., no tumor recurrence) to the patient for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 months or more after administration of the vaccine. In some embodiments, administering a nucleic acid nanostructure vaccine to a former cancer patient provides a therapeutic benefit (e.g., no tumor recurrence) to the patient for 0-10 weeks, 1-10 weeks, 2-7 weeks, 2-5 weeks, 5-10 weeks, or 5-20 weeks after administration of the vaccine. In some embodiments, administering a nucleic acid nanostructure vaccine to a former cancer patient provides a therapeutic benefit (e.g., no tumor recurrence) to the patient for at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150, or 200 days after administration of the vaccine.

[0115] As used herein, the term “cancer” refers to a disease state in which a population of cells in a subject has an abnormal ability to grow or replicate autonomously. In some embodiments, cancer is an abnormal state or condition characterized by proliferative cell growth (e.g., an abnormal state or condition of a tissue or organ). Cancer can include any solid or liquid, benign or malignant, non-invasive or invasive cancer or tumor, including growths, neoplasms, carcinomas, sarcomas, hematopoietic neoplastic disorders (e.g., leukemia), and pre-cancerous or pre-malignant lesions. A subject having cancer may exhibit symptoms such as a mass, abnormal bleeding, a persistent cough, unexplained weight loss, changes in bowel habits, gene mutations, fusion genes, and changes in chromosome number. In some embodiments, cancer is colorectal cancer, prostate cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, melanoma, bladder cancer, non-Hodgkin lymphoma, thyroid cancer, or brain cancer.

[0116] As used herein, the term “infectious disease” refers to a disease state caused by an exogenous organism (e.g., a microorganism) that enters a subject, multiplies, and causes a response in the subject (e.g., an inflammatory response). In some embodiments, an infectious disease is caused by a virus (or viral particle), bacterium, or fungus.

[0117] In some embodiments, provided herein are combination methods of treatment. In such combination methods, a nucleic acid nanostructure vaccine of the present disclosure and a second therapeutic agent are administered to a subject. In some embodiments, the second therapeutic agent is an anti-cancer drug. The anti-cancer drug can include checkpoint inhibitors (e.g., anti-PD-1, anti-PD-L1, anti-CTLA4, anti-TIM-3, anti-LAG-3); targeted kinase inhibitors (e.g., Imatinib mesylate, Ibrutinib, Neratinib, Palpociclib, Erlotinib, Lapatinib); antibodies (e.g., Bevacizumab, Trastuzumab, Rituximab, Cetuximab); chemotherapeutic agents such as deoxycytidine, pyrimidine or purine analogs (e.g., Irinotecan, 5-Fluorouracil, Lenalidomide, Capecitabine, Docetaxel); antibody-drug conjugates (e.g., Trastuzumab emtansine conjugate) or any other anti-cancer drug known to those of ordinary skill in the art. In some embodiments, the second therapeutic agent is an anti-PD-L1 antibody.

[0118] In some embodiments, the present disclosure provides methods for directly manipulating dendritic cell recruitment and activation in vivo. Immature dendritic cells patrol peripheral tissues and, upon uptake of foreign substances (e.g., antigens), they can mature to express molecules (e.g., the receptor CCR7 and major histocompatibility complex (MHC) antigens) on their surface, thereby promoting lymph node homing and subsequent antigen presentation to T cells, respectively. Infection factors that mobilize and activate dendritic cells include inflammatory cytokines and "danger signals" that are particularly associated with infectious agents. Cytosine-guanosine oligonucleotide (CpG-ODN) sequences are uniquely expressed in bacterial DNA and are potent danger signals that stimulate mammalian dendritic cell activation and dendritic cell migration. Thus, in some embodiments, the present disclosure provides methods for administering to a subject a nucleic acid nanostructure comprising an antigen (e.g., a cancer antigen) and a danger signal (e.g., a CpG oligonucleotide).

[0119] The "subjects" to be covered for administration include, but are not limited to, humans (e.g., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults or the elderly)) and / or other non-human animals, such as mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), including commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats and / or dogs), avians (e.g., commercially relevant avians such as chickens, ducks, geese and / or turkeys), reptiles, amphibians and fish. In some embodiments, the non-human animal is a mammal. The non-human animal can be male or female and can be at any stage of development. The non-human animal may be a transgenic animal.

[0120] The nucleic acid nanostructures and compositions containing the nucleic acid nanostructures can be administered to a subject (e.g., a human or non-human subject) by intratumoral, intramuscular, subcutaneous, intravenous (e.g., single / multiple injections or continuous infusion) or by other means.

[0121] In some embodiments, the nucleic acid nanostructures are administered as a component of a polymeric gel composition. The polymeric gel composition can be biocompatible and / or biodegradable. In some embodiments, the polymeric gel composition is formed of and / or contains at least one of the following: polylactic acid, polyglycolic acid, PLGA polymers, alginic acid and alginic acid derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids (e.g., polypeptides, especially polylysine), polyesters (e.g., polyhydroxybutyrate and polycaprolactone), polyanhydrides; polyphosphazenes, polyvinyl alcohol, polyalkylene oxides, especially polyethylene oxide, polyallylamine (PAM), polyacrylates, modified styrene polymers such as poly(4-aminomethylstyrene), Pluronic polyols, polyoxamers, glycuronans, polyvinylpyrrolidone and copolymers of the above, including graft copolymers (see, e.g., International Publication No. WO2009102465).

[0122] The nucleic acid nanostructure vaccine is administered in an effective amount to provide a therapeutic benefit (e.g., treatment, e.g., prevention of tumor formation or reduction of tumor volume) without causing excessive adverse reactions. The dose of the nucleic acid nanostructure vaccine required to achieve a particular therapeutic benefit will vary depending on a variety of factors, including but not limited to: the route of administration, the particular disease or disorder being treated (e.g., a particular type of cancer), and the stability of the vaccine (or combination therapy). Those skilled in the art can readily determine the dose of the nucleic acid nanostructure vaccine required and necessary to achieve a particular therapeutic benefit in a subject suffering from a particular disease or disorder based on the above factors and other factors well known in the art.

[0123] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to administer the nucleic acid nanostructure vaccines of the present disclosure into suitable host cells. The formation and use of liposomes are well known to those skilled in the art. Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multi-layer concentric bilayer vesicles (also known as multi-lamellar vesicles (MLV)). The diameter of MLV is typically 25 nm to 4 μm. Sonication of MLV results in the formation of small unilamellar vesicles (SUV) with a diameter ranging from 200 to that contain an aqueous solution in their core.

[0124] Other embodiments

[0125] Additional embodiments of the present disclosure are provided in the following numbered paragraphs:

[0126] 1. A nucleic acid barrel nanostructure conjugated with one or more antigen molecules and one or more adjuvant molecules.

[0127] 2. The nucleic acid barrel nanostructure of paragraph 1, wherein the distance between any two adjacent adjuvant molecules is 2 nm to 10 nm.

[0128] 3. The nucleic acid barrel nanostructure of paragraph 2, wherein the distance between any two adjacent adjuvant molecules is 4 - 10 nm, optionally 4 - 5 nm, 4 - 6 nm, 4 - 7 nm or 4 - 8 nm.

[0129] 4. The nucleic acid barrel nanostructure of paragraph 3 or 4, wherein the distance between any two adjacent adjuvant molecules is about 4.3 nm.

[0130] 5. The nucleic acid barrel nanostructure of any of the preceding paragraphs, wherein the density of adjuvant molecules on the nucleic acid nanostructure is 1 molecule per 5 to 50 nm 2 .

[0131] 6. The nucleic acid barrel nanostructure of paragraph 5, wherein the density of adjuvant molecules on the nucleic acid nanostructure is 1 molecule per 10 to 20 nm 2 or 1 molecule per 20 to 30 nm 2 .

[0132] 7. The nucleic acid barrel nanostructure of any of the preceding paragraphs, wherein the antigen molecules and / or adjuvant molecules are located on the surface of the nucleic acid nanostructure.

[0133] 8. The nucleic acid barrel nanostructure of any of the preceding paragraphs, which contains 5 to 25, 10 to 25 or 15 to 25 adjuvant molecules.

[0134] 9. The nucleic acid barrel nanostructure of any of the preceding paragraphs, wherein the antigen molecule is covalently conjugated to the nanostructure.

[0135] 10. The nucleic acid barrel nanostructure of any of the preceding paragraphs, which comprises DNA, RNA, or a mixture of DNA and RNA.

[0136] 11. The nucleic acid barrel nanostructure of paragraph 10, wherein the nucleic acid of the nanostructure comprises DNA or consists of DNA.

[0137] 12. A method comprising administering to a subject, in an effective amount, the nucleic acid barrel nanostructure of any of the preceding paragraphs to generate a T cell immune response in the subject.

[0138] 13. The method of paragraph 12, wherein the subject has a tumor.

[0139] 14. The method of paragraph 13, wherein the antigen molecule is a tumor antigen molecule.

[0140] 15. The method of any of the preceding paragraphs, wherein administration of the nanostructure stimulates a stronger Th1-type immune response relative to stimulating a Th2-type response.

[0141] 16. The method of any of the preceding paragraphs, wherein the administration is intratumoral, subcutaneous, intramuscular, or intravenous administration. Examples

[0142] Previously, DNA origami-based cubic lattice blocks (SQBs) loaded with antigens have been successfully assembled, with adjuvant spacing controlled. Through in vitro immune cell culture studies and in vivo tumor treatment models, data indicate that when the CpG spacing is 3.5 nm, the cubic blocks induce Th1-type immune polarization. This DNA origami cancer vaccine at low doses enhances antigen cross-presentation, CD8 + T cell activation, and CD4 + T cell activation with Th1-type polarization. The vaccine also synergizes with anti-PD-L1 for effective cancer regression in both melanoma and lymphoma. See International Publication No. WO 2020 / 247724, the entire content of which is incorporated herein.

[0143] Example 1: Basic barrel structure design and modification and folding optimization

[0144] The barrel structure as Figures 2A - 2B shown was designed to generate a vaccine platform. The barrel structure was folded using a bacteriophage M13 single-stranded DNA scaffold (7308 nt) complementary to hundreds of staple strands. Some small scaffolds were also applied in this design. All designs were done through honeycomb Cadnano software. There are three layers of double helices ( Figure 1), i.e., the outer layer (14 double helices), the middle layer, and the inner layer (15 double helices). Under the buffer conditions of 1×TE and 12 mM MgCl2, the barrels were self-assembled. Based on the basic barrel structure, we designed different modification sites for CpG, dsRNA, dyes, and coiled-coil peptides ( Figures 2A - 2B ). We also determined the folding conditions, purification conditions, and payload modification conditions of the barrel structure (Figures 3A - 3C). In some embodiments, the temperature gradient of the barrel was denatured at 80 degrees for 15 minutes and then decreased from 65 degrees to 25 degrees within 18 hours. Exemplary barrel purification conditions were that the final mixture contained a 5% PEG solution, the barrel and the 10% PEG solution were mixed at a volume ratio of 1:1, and MgCl2 was maintained at 12 mM. For CpG and dsRNA conjugation, the adjuvant was added in a two-fold excess, which provided complete conjugation efficiency. Barrel production was efficient, reproducible, and scalable.

[0145] Example 2: Construction of the Barrel Vaccine Platform

[0146] We successfully constructed the adjuvants CpG and dsRNA (TLR3 ligand) on the barrel, where the spacing between adjacent adjuvant molecules was 4.3 nm ( Figures 4A - 4D ). By co-culturing the barrel-shaped origami constructed with CpG and dsRNA with HEK blue cells expressing TLR9 (CpG receptor) or TLR3 (dsRNA receptor), we detected an increase in receptor activation compared to free adjuvants ( Figures 5A - 5B ). 1 nM barrels conjugated with (1) both CpG and dsRNA (“barrel-CpG-dsRNA”), (2) only CpG (“barrel-CpG”), or (3) only dsRNA (“barrel-dsRNA”) were used for cell stimulation within 24 hours. The corresponding concentrations of CpG and dsRNA were 24 nM. In the free adjuvant control (i.e., the adjuvant was not conjugated to the barrel), 24 nM CpG (“CpG”) or 24 nM dsRNA (“dsRNA”) was used. A negative control (no adjuvant or barrel, “NC”) was also tested.

[0147] In addition, we identified several candidate coiled-coil peptides (CCPs), namely E4_N, K4_N, K / E4_N, E4_C, and E3_N, as potential neoantigen capture motifs (Figure 6A). N or C refers to the terminus linked to the 42 DNA handle regions as shown in Figure 2C. These peptides form stable α-helices with hydrophilic and hydrophobic surfaces. The hydrophobic surface readily binds / interacts with the hydrophobic surfaces of peptides or proteins. We have successfully conjugated CCPs that capture neoantigens inside the barrel to complete the DNA origami antigen capture platform (Figures 6B-6E). E4_N, K / E4_N, and E4_C performed well during construction (Figure 6D). TEM results confirmed the successful conjugation of all components (Figure 6E). Gel analysis confirmed that once the coiled-coil peptides were attached inside the barrel, they could capture excess coiled-coil peptides, with increasing CCP-conjugated barrels as more excess (0.5 to 4-fold) was added (Figures 6F, 6G). These results provide validation for the barrel vaccine platform.

[0148] Example 3: Short Peptides Captured by Barrel Vaccine Nanoparticles

[0149] Next, we tested the ability of different coiled-coil peptides (CCPs) to capture short peptides with different hydrophobicities. The barrel-shaped origami was conjugated with E4_N, E4_C, or E4 / K4_N CCPs and exposed to short peptide sequences (FGFGF, RGFGY, or GGFGG). Overall, the hydrophobicity level was FGFGF > RGFGY > GGFGG. Our results showed that both CCP E4_N and CCP E4_C could capture more hydrophobic FGFGF peptides compared to other peptides (Figures 7A-7B). E4 / K4_N captured few peptides, probably due to its neutral charge. We also tested the supernatant after the barrel CCPs captured short peptides and were precipitated from the solution with PEG. HPLC results showed that the concentration of peptides remaining in the supernatant increased while the hydrophobicity of the peptides decreased (Figure 7C). These data indicate that coiled-coil peptides are capable of preferentially capturing hydrophobic peptides.

[0150] Example 4: The Barrel Vaccine Platform Successfully Captured Proteins Released from Irradiated Tumor Cells

[0151] To demonstrate whether the barrel-shaped vaccine can capture proteins released from tumor cells after immunogenic cell death (ICD), we irradiated CT26 colon cancer and B16F10 melanoma tumor cell lines with 100 Gy of photons and concentrated the culture supernatant by MWCO filtration (Figure 8A - 8C). We co-cultured the barrel-shaped vaccine with the supernatant. Subsequently, we purified the barrels by PEG and then applied DNase I for digestion. DNase I digestion will remove the barrel structure but retain the captured proteins for detection. The protocol for this example is shown in Figure 8A. We ran the samples on an SDS PAGE gel and performed silver staining to verify that barrel-E4_N can successfully capture the proteins secreted by the irradiated cells (Figure 8D). These results indicate that CCP can capture antigens from tumor ICD.

[0152] Example 5: Mass spectrometry analysis of the captured proteins

[0153] We have confirmed that the barrel structure (conjugated with coiled-coil peptides) can capture proteins secreted by irradiated cells. Next, we used mass spectrometry to understand what antigens were captured. For B16F10 and CD26, we found that the barrel structure captured several tumor neoantigens (Figure 9A - 9B). Specifically, in the B16F10 model, the barrel structure (conjugated with coiled-coil peptides) captured Actn4, Eef2, Tubb6, Tubb3, Plod1, and Got2 (Figure 9A); in the CT26 model, the barrel structure (conjugated with coiled-coil peptides) captured Actbl2, Septin7, and Fn1. We also identified heat shock proteins and other DAMP-related proteins highly enriched in the proteins captured by the barrel structure (Figure 9C - 9D), such as Hspa5, Hspa8, Hsp90b1, Hsp90aa1, Hmgb1, and H4f16. In a more refined exploration of B16F10, we compared the protein abundances under different barrel conditions and verified that the antigen capture efficiency was the highest only when CCP was included in the barrel (Figure 9E - 9F).

[0154] Example 6: The barrel-shaped DNA origami vaccine (DoriVac) shows efficacy against highly resistant tumor models

[0155] The results in this example show that in the highly resistant B16F10 melanoma model and another colon cancer model, the combination of the antigen-capturing barrel-shaped DoriVac and the therapeutically effective anti-PD-L1 antibody promoted robust and durable tumor control (Figure 10 - Figure 14).

[0156] To use the barrel-shaped DoriVac as a complete vaccine, the vaccine construct was completed before application to the subjects (mouse subjects). First, the barrel was conjugated with an adjuvant and an antigen-capturing motif (e.g., a coiled-coil peptide). Then the barrel was exposed to the antigen to allow the barrel to capture the antigen, which was verified by silver staining as described previously. A B16F10 tumor model was established by injecting 200K or 100K tumor cells into the right flank of the mice. On days 3, 7, and 13 after tumor inoculation, vaccine treatment (barrel-shaped DoriVac or bolus (tumor supernatant, free CCP, and free CpG and dsRNA adjuvant (without barrel-shaped nanostructure))) was administered subcutaneously at the left shoulder, and on days 6, 8, 10, 12, 14, and 16, anti-PD-L1 was administered subcutaneously at the same site as the vaccine administration (Figure 10A, Figure 12A). The tumors and survival of the mice were recorded.

[0157] It was found that the combination treatment of barrel-shaped Dorivac and anti-PD-L1 antibody could significantly inhibit tumor growth and prolong the survival of mice (Figure 10B - 10C, Figure 12B - 12C).

[0158] In mice given 200k tumor cells, the tumor volume of the mice receiving the combination treatment of barrel-shaped DoriVac and anti-PD-L1 antibody was significantly reduced ( Figure 10C ) and their lifespan was prolonged (mice selected to receive barrel-shaped DoriVac and anti-PD-L1 antibody treatment were able to survive to at least day 40, while all mice in other treatment groups died before day 30) ( Figure 10D ). For the 200K B16F10 model, we also determined the immune cell lineage typing of the mice. The data showed that the PD-L1 + cell level was reduced (PD-L1 inhibition) in mice receiving the combination treatment of barrel-shaped DoriVac and anti-PD-L1 antibody compared to mice receiving only barrel-shaped DoriVac treatment (Figure 11B). Enhanced CD4 and CD8 T cell activation was also shown by IFNγ expression in mice receiving combination treatment (Figure 11E - 11F).

[0159] When we used a less invasive B16F10 tumor model (100K cells) (Figure 12A), both the single barrel-shaped DoriVac and the combination of barrel-shaped DoriVac and anti-PD-L1 antibody also significantly reduced the tumor volume ( Figure 12C ) and prolonged the lifespan (3 out of 7 mice in each treatment group survived to day 50 at the end of the experiment) ( Figure 12D ).

[0160] In a B16F10 tumor model (100K cells), mice that survived 50 days after receiving DoriVac or DoriVac / anti-PD-L1 antibody treatment (n = 3 per treatment group) were challenged with a fresh inoculation of 1x10 5 B16F10 tumor cells. Mice that received the initial inoculation of the same number of cells were used as the control group. As Figure 12E shown, all mice in both treatment groups survived approximately 25 days after tumor rechallenge, and two mice in each treatment group survived for more than 40 days.

[0161] These data indicate that nucleic acid barrel nanostructure vaccines as described herein (e.g., conjugated with adjuvant molecules and antigen capture motifs) can shrink tumor volume and treat cancer (in the test model) by prolonging the lifespan. These beneficial effects can be demonstrated when the nucleic acid barrel nanostructure vaccine is used alone or in combination with another cancer treatment molecule (e.g., anti-PD-L1 antibody). Moreover, the data from tumor rechallenge indicate that nucleic acid barrel nanostructure vaccines can provide functional benefits to subjects for treating the initial tumor and preventing tumor recurrence.

[0162] We also sought to understand whether barrel-shaped DoriVac could locally capture antigens after chemotherapy drug-induced immunogenic cell death (ICD) (Figure 13). Six days after tumor inoculation (500K B16F10 cells), doxorubicin was applied when the tumors reached 50 - 80 mm 2 , and a barrel-shaped vaccine delivery platform was applied intratumorally after ICD. Anti-PD-L1 was applied to the area surrounding the tumor tissue (Figure 13A). We could observe the synergistic effect of doxorubicin ICD and the barrel-shaped vaccine delivery platform (Figure 13B - 13C). Relative to untreated mice exposed to doxorubicin, mice exposed to doxorubicin and subsequently treated with barrel-shaped DoriVac (“barrel-shaped ISV”) or a combination of barrel-shaped DoriVac and anti-PD-L1 antibody (“barrel-shaped ISV + αPD-L1”) had prolonged survival rates ( Figure 13C ). Several mice survived until the end of the experiment (at 50 days). Mice that survived 50 days after receiving DoriVac or DoriVac / anti-PD-L1 antibody treatment were challenged with a fresh inoculation of 1x10 5 B16F10 tumor cells. Mice that received the initial inoculation of the same number of cells were used as the control group. As Figure 13D shown, mice treated with DoriVac exhibited prolonged survival rates, with several mice in the DoriVac treatment surviving approximately 70 days after tumor rechallenge.

[0163] The efficacy of barrel-shaped DoriVac was also tested in the MC38 colon cancer model. The MC38 tumor model was established by injecting 200K tumor cells into the right flank of mice. On days 3, 7, and 13 after tumor inoculation, vaccine treatment (barrel-shaped DoriVac or bolus (without barrel structure)) was administered subcutaneously at the left shoulder, and on days 6, 8, 10, 12, 14, and 16, anti-PD-L1 was administered subcutaneously at the same site as the vaccine administration (Figure 14A). Mouse tumors and survival were recorded. Treatment with barrel-shaped DoriVac together with anti-PD-L1 cured 5 out of 8 mice and showed an extended survival period of the mice ( Figure 14C ). Mice that survived 50 days after treatment with DoriVac / anti-PD-L1 antibody or bolus vaccine / anti-PD-L1 antibody were challenged with a fresh inoculation of 2x10 5 MC38 tumor cells. The initial mice that received the same amount of cells were used as the control group. As Figure 14D shown, none of the mice treated with DoriVac died due to the rechallenge, demonstrating the durable function of this vaccine.

[0164] Example 7: Barrel-shaped DNA origami vaccine (DoriVac) showed efficacy as a prophylactic vaccine

[0165] Barrel-shaped DoriVac (a nucleic acid barrel nanostructure with a coiled-coil peptide with antigen capture ability and an adjuvant) was prepared as described in the previous Example 6. C57BL6 mice received two doses of DoriVac, bolus vaccine (tumor supernatant, free CCP, free CpG, and dsRNA adjuvant), or saline (control) on days 0 and 7. On day 7, 1x10 5 B16F10 cells or 2x10 5 MC38 cells were inoculated into the right flank of the mice. Mouse survival was recorded (n = 5).

[0166] As Figure 15A shown, all mice that received prophylactic treatment with DoriVac vaccine before challenge with B16F10 tumor cells survived until the end of the experimental period (day 80). Among the mice treated with bolus vaccine, less than 50% survived until the end of the experimental period; and none of the untreated control mice survived beyond day 40.

[0167] As Figure 15B shown, all mice that received prophylactic treatment with DoriVac vaccine before challenge with MC38 tumor cells survived until the end of the experimental period (day 80). Among the mice treated with bolus vaccine, less than 50% survived until the end of the experimental period; and none of the untreated control mice survived beyond day 50.

[0168] These data indicate that prophylactic administration (i.e., administration before cancer onset) of nucleic acid barrel nanostructure vaccines as described herein (e.g., conjugated with adjuvant molecules and antigen capture motifs) can be used to treat cancer (by extending the lifespan). Specifically, relative to cancer vaccines without nucleic acid barrel nanostructures, prophylactic administration of nucleic acid barrel nanostructure vaccines as described herein can extend the average lifespan of subjects with cancer.

[0169] All references, patents, and patent applications disclosed herein are incorporated by reference into the respective subject matter they are cited in, where in some cases, such subject matter may include the entire contents of the document.

[0170] As used in the specification and claims herein, the indefinite articles "a" and "an" shall be understood to mean "at least one" unless expressly stated to the contrary.

[0171] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the recited order of the steps or acts of the method.

[0172] In the claims and the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," etc., shall be understood to be open-ended, i.e., meaning including but not limited to. According to the provisions of Section 2111.03 of the Manual of Patent Examining Procedure of the United States Patent and Trademark Office, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively.

[0173] The terms "about" and "substantially" before a numerical value indicate ±10% of the stated numerical value.

[0174] Where a range of values is provided, each value between the upper and lower limits of that range is specifically covered and described herein.

Claims

1. A nucleic acid barrel-shaped nanostructure conjugated with an adjuvant molecule and an antigen-capturing motif.

2. A nucleic acid barrel-shaped nanostructure conjugated with an antigen molecule and an adjuvant molecule.

3. The nucleic acid barrel-shaped nanostructure according to claim 2, further comprising an antigen-capturing motif.

4. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the distance between any two adjacent adjuvant molecules is from 2 nm to 10 nm.

5. The nucleic acid barrel-shaped nanostructure according to claim 4, wherein the distance between any two adjacent adjuvant molecules is from 4 - 10 nm, optionally from 4 - 5 nm, 4 - 6 nm, 4 - 7 nm or 4 - 8 nm.

6. The nucleic acid barrel-shaped nanostructure according to claim 4 or 5, wherein the distance between any two adjacent adjuvant molecules is about 4.3 nm.

7. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the density of adjuvant molecules on the nucleic acid nanostructure is from 1 molecule per 5 nm 2 to 1 molecule per 50 nm 2 .

8. The nucleic acid barrel-shaped nanostructure according to claim 7, wherein the density of adjuvant molecules on the nucleic acid nanostructure is 1 molecule per 10 to 20 nm 2 or 1 molecule per 20 to 30 nm 2 and there is 1 molecule.

9. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the antigen molecule and / or the antigen-capturing motif and / or the adjuvant molecule are located on the surface of the nucleic acid nanostructure.

10. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the nanostructure comprises an adjuvant molecule conjugated to the outer surface of the nanostructure.

11. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the nanostructure comprises an antigen-capturing motif conjugated to the inner surface of the nanostructure.

12. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the nanostructure comprises an adjuvant molecule conjugated to the outer surface of the nanostructure and an antigen-capturing motif conjugated to the inner surface of the nanostructure.

13. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, which comprises 5 to 200, 100 to 200, 100 to 150, 5 to 25, 10 to 25 or 15 to 25 adjuvant molecules.

14. The nucleic acid barrel-shaped nanostructure according to any one of claims 2 - 13, wherein the antigen molecule is covalently conjugated to the nanostructure.

15. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, wherein the antigen-capturing motif is a coiled-coil peptide.

16. The nucleic acid barrel-shaped nanostructure according to claim 15, wherein the coiled-coil peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 - 10.

17. The nucleic acid barrel-shaped nanostructure according to any one of the preceding claims, which comprises DNA, RNA or a mixture of DNA and RNA.

18. The nucleic acid barrel-shaped nanostructure according to claim 17, wherein the nucleic acid of the nanostructure comprises DNA or consists of DNA.

19. A composition comprising the nucleic acid barrel-shaped nanostructure according to any one of the preceding claims and a second therapeutic agent, optionally wherein the second therapeutic agent is an anti-cancer drug, further optionally wherein the anti-cancer drug is an anti-PD-L1 antibody.

20. A pharmaceutical composition for vaccinating a subject against a disease, wherein the pharmaceutical composition comprises the nucleic acid barrel-shaped nanostructure according to any one of claims 1 - 18.

21. A method comprising administering to a subject, in an effective amount, the nucleic acid barrel nanostructure according to any one of claims 1-18, the composition according to claim 19, or the pharmaceutical composition according to claim 20, to generate a T cell immune response in the subject.

22. A method of treating a subject having a disease, the method comprising administering to the subject, in an effective amount, the nucleic acid barrel nanostructure according to any one of claims 1-18, the composition according to claim 19, or the pharmaceutical composition according to claim 20 to treat the disease.

23. The method according to claim 21 or 22, wherein the subject has a tumor, optionally a cancerous tumor.

24. The method according to claim 23, wherein the antigen molecule is a tumor antigen molecule.

25. The method according to any one of claims 21-24, wherein administration of the nanostructure stimulates a stronger Th1-type immune response relative to stimulation of a Th2-type response.

26. The method according to any one of claims 22-25, wherein the disease is cancer.

27. A method comprising administering to a subject the nucleic acid barrel nanostructure according to any one of claims 1-18, the composition according to claim 19, or the pharmaceutical composition according to claim 20.

28. The method according to claim 27, wherein the nucleic acid barrel nanostructure, composition or pharmaceutical composition is administered in an effective amount to prevent the onset of a disease, optionally 1, 2, 3, 4, 5, 6, 7, 8, 9 months or more months after said administration.

29. The method according to any one of claims 19-28, wherein the administration is intratumoral, subcutaneous or intravenous administration.

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