Chemically Tuning the Antigen Release Kinetics from Spherical Nucleic Acids Maximizes Immune Stimulation.

Cancer vaccine structure is emerging as an important design factor that offers tunable parameters to enhance the targeted immune response. We report the impact of altering the antigen release rate from spherical nucleic acid (SNA) vaccines-nanoparticles with a liposomal core and surface-anchored adjuvant DNA-on immune stimulation. Peptide antigens were incorporated into SNAs using either a nonreducible linker or one of a series of reduction-triggered traceless linkers that release the native peptide at rates controlled by their substitution pattern. Compared with a nonreducible linkage, the traceless attachment of antigens resulted in lower EC50 of T cell proliferation in vitro and greater dendritic cell (DC) activation and higher T cell killing ability in vivo. Traceless linker fragmentation rates affected the rates of antigen presentation by DCs and were correlated with the in vitro potencies of SNAs. Antigen release was correlated with the ex vivo -log(EC50), and more rapid antigen release resulted in an order of magnitude improvement in the EC50 and earlier and greater antigen presentation over the same time-period. In vivo, increasing the rate of antigen release resulted in higher T cell activation and target killing. These findings provide fundamental insights into and underscore the importance of vaccine structure.

Development of Spherical Nucleic Acids for Prostate Cancer Immunotherapy.

Although the strategy of therapeutic vaccination for the treatment of prostate cancer has advanced to and is available in the clinic (Sipuleucel-T), the efficacy of such therapy remains limited. Here, we develop Immunostimulatory Spherical Nucleic Acid (IS-SNA) nanostructures comprised of CpG oligonucleotides as adjuvant and prostate cancer peptide antigens, and evaluate their antitumor efficacy in syngeneic mouse models of prostate cancer. IS-SNAs with the specific structural feature of presenting both antigen and adjuvant CpG on the surface (hybridized model (HM) SNAs) induce stronger cytotoxic T lymphocyte (CTL) mediated antigen-specific killing of target cells than that for IS-SNAs with CpG on the surface and antigen encapsulated within the core (encapsulated model (EM) SNAs). Mechanistically, HM SNAs increase the co-delivery of CpG and antigen to dendritic cells over that for EM SNAs or admixtures of linear CpG and peptide, thereby improving cross-priming of antitumor CD8+ T cells. As a result, vaccination with HM SNAs leads to more effective antitumor immune responses in two prostate cancer models. These data demonstrate the importance of the structural positioning of peptide antigens together with adjuvants within IS-SNAs to the efficacy of IS-SNA-based cancer immunotherapy.

Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides.

This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-µmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMadeTM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMadeTM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.

Light-Responsive Colloidal Crystals Engineered with DNA.

A novel method for synthesizing and photopatterning colloidal crystals via light-responsive DNA is developed. These crystals are composed of 10-30 nm gold nanoparticles interconnected with azobenzene-modified DNA strands. The photoisomerization of the azobenzene molecules leads to reversible assembly and disassembly of the base-centered cubic (bcc) and face-centered cubic (fcc) crystalline nanoparticle lattices. In addition, UV light is used as a trigger to selectively remove nanoparticles on centimeter-scale thin films of colloidal crystals, allowing them to be photopatterned into preconceived shapes. The design of the azobenzene-modified linking DNA is critical and involves complementary strands, with azobenzene moieties deliberately staggered between the bases that define the complementary code. This results in a tunable wavelength-dependent melting temperature (Tm ) window (4.5-15 °C) and one suitable for affecting the desired transformations. In addition to the isomeric state of the azobenzene groups, the size of the particles can be used to modulate the Tm window over which these structures are light-responsive.

Mercury-Free Automated Synthesis of Guanidinium Backbone Oligonucleotides.

A new method for synthesizing deoxynucleic guanidine (DNG) oligonucleotides that uses iodine as a mild and inexpensive coupling reagent is reported. This method eliminates the need for the toxic mercury salts and pungent thiophenol historically used in methods aimed at preparing DNG oligonucleotides. This coupling strategy was readily translated to a standard MerMade 12 oligonucleotide synthesizer with coupling yields of 95% and has enabled the synthesis of a 20-mer DNG oligonucleotide, the longest DNG strand to date, in addition to mixed DNA-DNG sequences with 3-9 DNG inserts. Importantly, DNG oligonucleotides exhibit robust unaided cellular uptake as compared to unmodified oligonucleotides without apparent cellular toxicity. Taken together, these findings should greatly increase the accessibility of cationic backbone modifications and assist in the development of oligonucleotide-based drugs.

Rational vaccinology with spherical nucleic acids.

In the case of cancer immunotherapy, nanostructures are attractive because they can carry all of the necessary components of a vaccine, including both antigen and adjuvant. Herein, we explore how spherical nucleic acids (SNAs), an emerging class of nanotherapeutic materials, can be used to deliver peptide antigens and nucleic acid adjuvants to raise immune responses that kill cancer cells, reduce (or eliminate) tumor growth, and extend life in three established mouse tumor models. Three SNA structures that are compositionally nearly identical but structurally different markedly vary in their abilities to cross-prime antigen-specific CD8+ T cells and raise subsequent antitumor immune responses. Importantly, the most effective structure is the one that exhibits synchronization of maximum antigen presentation and costimulatory marker expression. In the human papillomavirus-associated TC-1 model, vaccination with this structure improved overall survival, induced the complete elimination of tumors from 30% of the mice, and conferred curative protection from tumor rechallenges, consistent with immunological memory not otherwise achievable. The antitumor effect of SNA vaccination is dependent on the method of antigen incorporation within the SNA structure, underscoring the modularity of this class of nanostructures and the potential for the deliberate design of new vaccines, thereby defining a type of rational cancer vaccinology.

Conjugation Chemistry-Dependent T-Cell Activation with Spherical Nucleic Acids.

Spherical nucleic acids (SNAs) can be potent sequence-specific stimulators of antigen presenting cells (APCs). When loaded with peptide antigens, they can be used to activate the immune system to train T-cells to specifically kill cancer cells. Herein, the role of peptide chemical conjugation to the DNA, which is used to load SNAs with antigens via hybridization, is explored in the context of APC activation. Importantly, though the antigen chemistry does not impede TLR-9 regulated APC activation, it significantly augments the downstream T-cell response in terms of both activation and proliferation. A comparison of three linker types, (1) noncleavable, (2) cleavable but nontraceless, and (3) traceless, reveals up to an 8-fold improvement in T-cell proliferation when the traceless linker is used. This work underscores the critical importance of the choice of conjugation chemistry in vaccine development.

Catalyst-controlled Wacker-type oxidation: facile access to functionalized aldehydes.

The aldehyde-selective oxidation of alkenes bearing diverse oxygen groups in the allylic and homoallylic position was accomplished with a nitrite-modified Wacker oxidation. Readily available oxygenated alkenes were oxidized in up to 88% aldehyde yield and as high as 97% aldehyde selectivity. The aldehyde-selective oxidation enabled the rapid, enantioselective synthesis of an important pharmaceutical agent, atomoxetine. Finally, the influence of proximal functional groups on this anti-Markovnikov reaction was explored, providing important preliminary mechanistic insight.