Opportunities and challenges for the clinical translation of structured DNA assemblies as gene therapeutic delivery and vaccine vectors

Abstract

Gene therapeutics including siRNAs, anti-sense oligos, messenger RNAs, and CRISPR ribonucleoprotein complexes offer unmet potential to treat over 7,000 known genetic diseases, as well as cancer, through targeted in vivo modulation of aberrant gene expression and immune cell activation. Compared with viral vectors, nonviral delivery vectors offer controlled immunogenicity and low manufacturing cost, yet suffer from limitations in toxicity, targeting, and transduction efficiency. Structured DNA assemblies fabricated using the principle of scaffolded DNA origami offer a new nonviral delivery vector with intrinsic, yet controllable immunostimulatory properties and virus-like spatial presentation of ligands and immunogens for cell-specific targeting, activation, and control over intracellular trafficking, in addition to low manufacturing cost. However, the relative utilities and limitations of these vectors must clearly be demonstrated in preclinical studies for their clinical potential to be realized. Here, we review the major capabilities, opportunities, and challenges we foresee in translating these next-generation delivery and vaccine vectors to the clinic. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Keywords: DNA origami; gene therapeutic; nonviral delivery vector; structured DNA assemblies; vaccine.

FIGURE 1

Structure‐composition comparison of AAV9 and virus‐like DNA assemblies fabricated using the scaffolded DNA origami approach. (a) Cryo‐EM density map of the icosahedral AAV9 capsid with an octant removed to show the capsid interior. The capsid consists of 60 self‐assembled copies total of viral proteins (VP) VP1, VP2, and VP3 in a ratio of 1:1:10, respectively. (b) Pseudoatomic model of AAV9 using AAV2 and AAV8 crystal structures as templates, showing the surface loops VR‐I and VR‐IV that control transduction phenotype and help determine tropism and antigenic reactivity in addition to transduction efficiency (DiMattia et al., 2012). The ssDNA AAV genome is approximately 4.8 kb and the AAV capsid is approximately 25 nm in diameter. (c) Cryo‐EM density map of an icosahedral DNA origami designed using two crosslinked duplexes per edge, where each edge is 52 basepairs or 18 nm long (Veneziano et al., 2016). The 3D atomic model generated by the automatic sequence design algorithm DAEDALUS is shown superposed on the EM density. The geometrically virus‐like particle is approximately 40 nm in diameter and contains a ssDNA scaffold of 3,120 bases. (d) Cryo‐EM density map of an octahedral DNA origami consisting of six crosslinked duplexes per edge, where each edge is 84 basepairs or 29 nm long (Jun, Shepherd, et al., 2019). The overall particle is approximately 41 nm in diameter and contains a ssDNA scaffold of 6,762 bases. Panels a and b are reproduced by permission from DiMattia et al. (2012). Panel c is reproduced by permission from Veneziano et al. (2016). Panel d is reproduced by permission from Jun, Shepherd, et al. (2019)

FIGURE 2

Formulation and synthesis of virus‐like structured DNA assemblies. (a) Fully automated design software DAEDALUS (Veneziano et al., 2016) for two‐helix bundle edges or TALOS (Jun, Shepherd, et al., 2019) for six‐helix bundle edges is used to route the ssDNA scaffold and complementary oligonucleotide staples that hybridize to the scaffold to self‐assemble the target virus‐like geometry. Any polyhedral geometry can be fabricated with edge lengths specified between ~10 and 50 nm. (b) Scalable biological production of custom sequence and length ssDNA staples can be achieved in Escherichia coli using an M13 phagemid and a helper phage or (c) ssDNA scaffold using a helper plasmid such as M13cp (Chasteen, Ayriss, Pavlik, & Bradbury, 2006) grown in a commercial bioreactor (Praetorius et al., 2017; Shepherd, Du, Huang, Wamhoff, & Bathe, 2019). Fixed scaffold sequences can be eliminated using either a split‐origin of replication strategy (Nafisi, Aksel, & Douglas, 2018) or self‐cleaving DNazyme inserts shown in (b) that result in linear instead of circular ssDNA (Engelhardt et al., 2019; Praetorius et al., 2017). Staples are shown in (b) as different colors in the circular ssDNA at left, with Zn²⁺ added to activate the DNazyme self‐cleaving reaction, which then results in the linear staples with small overhangs that then self‐assemble into the brick‐like origami shown. (d) Synthetic oligonucleotide staples vary in length between 20 and 60 nucleotides with free 3′ and 5′ ends that can be functionalized using a variety of chemistries (Wamhoff et al., 2019). Alternatively, they can be designed with ~10–20 nucleotide ssDNA overhangs of prescribed sequences that protrude either into or out of the particle, which complementary ssDNA, ssPNA, ssLNA, or ssRNA can then hybridize to for noncovalent, reversible attachment of either nucleic acid, small molecule, protein, or peptide targeting ligands, external to the particle, or therapeutic cargo, internal to the particle. Panel a: Reprinted with permission from Veneziano et al. (2016). Copyright 2016 The American Association for the Advancement of Science. Panel b: Reprinted with permission from Praetorius et al. (2017). Copyright 2017 Springer Nature. Panel c: Reprinted with permission from Shepherd et al. (2019). Copyright 2019 Springer Nature. Panel d: Reprinted with permission from Wamhoff et al. (2019). Copyright 2019 Annual Reviews, Inc.

FIGURE 3

Therapeutic modalities with structured DNA assemblies. (a) Any virus‐like structured DNA assembly can be used to present ligands outwardly for immune cell stimulation, cell targeting, intracellular trafficking, or some combination of these. Thirty copies of the HIV envelope glycoprotein gp120 are shown conjugated to the outside of the icosahedral DNA origami with two‐helix bundle edges. (b) Instead of using a ssDNA scaffold to fold virus‐like DNA nanoparticles, messenger RNA scaffold can be used, wherein the structure shows the ~1 kb nucleotide mRNA from Green Fluorescent Protein folded into a pentagonal bipyramid by designing complementary ssDNA staples (Parsons et al., 2019). Staples could be functionalized with 3′ and 5′ targeting or other moieties, as in (a). (c) Alternatively, the mRNA, in this case RFP, can be co‐formulated to hybridize to the interior of the fully DNA origami nanoparticle, in this case a simple tetrahedron with two‐helix bundle edges, by presenting inward facing ssDNA overhangs that are complementary to loop regions of the mRNA (Bathe lab, unpublished data). (d) Similarly, CRISPR RNPs can be co‐formulated with ssDNA HDR template by hybridizing the guide RNAs to complementary ssDNA overhangs on the interior of the pentagonal bipyramid structured DNA assembly shown, with multiple siRNAs, all of which can in principle be released in a programmed manner using pH‐ or receptor‐mediated triggering mechanisms