Explore the vast range of titles available.

The increasing number of approved nucleic acid therapeutics demonstrates the potential to treat diseases by targeting their genetic blueprints in vivo. Conventional treatments generally induce therapeutic effects that are transient because they target proteins rather than underlying causes. In contrast, nucleic acid therapeutics can achieve long-lasting or even curative effects via gene inhibition, addition, replacement or editing. Their clinical translation, however, depends on delivery technologies that improve stability, facilitate internalization and increase target affinity. Here, we review four platform technologies that have enabled the clinical translation of nucleic acid therapeutics: antisense oligonucleotides, ligand-modified small interfering RNA conjugates, lipid nanoparticles and adeno-associated virus vectors. For each platform, we discuss the current state-of-the-art clinical approaches, explain the rationale behind its development, highlight technological aspects that facilitated clinical translation and provide an example of a clinically relevant genetic drug. In addition, we discuss how these technologies enable the development of cutting-edge genetic drugs, such as tissue-specific nucleic acid bioconjugates, messenger RNA and gene-editing therapeutics.This Review provides an overview of four platform technologies that are currently used in the clinic for delivery of nucleic acid therapeutics, describing their properties, discussing technical advancements that led to clinical approval, and highlighting examples of approved genetic drugs that make use of these technologies.

In humans, lipid nanoparticles (LNPs) have safely delivered therapeutic RNA to hepatocytes after systemic administration and to antigen-presenting cells after intramuscular injection. However, systemic RNA delivery to non-hepatocytes remains challenging, especially without targeting ligands such as antibodies, peptides, or aptamers. Here we report that piperazine-containing ionizable lipids (Pi-Lipids) preferentially deliver mRNA to immune cells in vivo without targeting ligands. After synthesizing and characterizing Pi-Lipids, we use high-throughput DNA barcoding to quantify how 65 chemically distinct LNPs functionally delivered mRNA (i.e., mRNA translated into functional, gene-editing protein) in 14 cell types directly in vivo. By analyzing the relationships between lipid structure and cellular targeting, we identify lipid traits that increase delivery in vivo. In addition, we characterize Pi-A10, an LNP that preferentially delivers mRNA to the liver and splenic immune cells at the clinically relevant dose of 0.3 mg/kg. These data demonstrate that high-throughput in vivo studies can identify nanoparticles with natural non-hepatocyte tropism and support the hypothesis that lipids with bioactive small-molecule motifs can deliver mRNA in vivo. Next-generation lipid nanoparticles that target non-hepatocytes could be important clinical tools. Using in vivo DNA barcoding, the authors identify piperazine-containing lipids deliver mRNA to immune cells without targeting ligands.

We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications.

Immunotherapy with immune checkpoint blockade (ICB) has shown limited benefits in hepatocellular carcinoma (HCC) and other cancers, mediated in part by the immunosuppressive tumor microenvironment (TME). As p53 loss of function may play a role in immunosuppression, we herein examine the effects of restoring p53 expression on the immune TME and ICB efficacy. We develop and optimize a CXCR4-targeted mRNA nanoparticle platform to effectively induce p53 expression in HCC models. Using p53 -null orthotopic and ectopic models of murine HCC, we find that combining CXCR4-targeted p53 mRNA nanoparticles with anti-PD-1 therapy effectively induces global reprogramming of cellular and molecular components of the immune TME. This effect results in improved anti-tumor effects compared to anti-PD-1 therapy or therapeutic p53 expression alone. Thus, our findings demonstrate the reversal of immunosuppression in HCC by a p53 mRNA nanomedicine when combined with ICB and support the implementation of this strategy for cancer treatment. The p53 tumor suppressor gene is frequently mutated in liver cancer. Here the authors show that restoration of p53 expression with a mRNA nanoparticle platform elicits anti-tumor immune responses and promotes response to immune checkpoint blockade in preclinical models of p53-null hepatocellular carcinoma.

RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices. RNA nanostructures can be designed to fold during transcription, but the solution structure has remained elusive. Here the authors use cryogenic electron microscopy to determine the structure of a panel of RNA origami shapes and uncover the design and folding principles.

Molecular simulation has become an integral part of the DNA/RNA nanotechnology research pipeline. In particular, understanding the dynamics of structures and single-molecule events has improved the precision of nanoscaffolds and diagnostic tools. Here we present oxView, a design tool for visualization, design, editing and analysis of simulations of DNA, RNA and nucleic acid–protein nanostructures. oxView provides an accessible software platform for designing novel structures, tweaking existing designs, preparing them for simulation in the oxDNA/RNA molecular simulation engine and creating visualizations of simulation results. In several examples, we present procedures for using the tool, including its advanced features that couple the design capabilities with a coarse-grained simulation engine and scripting interface that can programmatically edit structures and facilitate design of complex structures from multiple substructures. These procedures provide a practical basis from which researchers, including experimentalists with limited computational experience, can integrate simulation and 3D visualization into their existing research programs.Sulc and colleagues describe a design tool, oxView, for visualization and analysis of simulations of DNA, RNA and nucleic acid–protein nanostructures.

RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology.RNA origami can be used for the modular design of RNA nanoscaffolds but can be challenging to design. Newly developed computer-aided design software has now been shown to improve the folding yield of kilobase-sized RNA origami. These structures fold from a single strand during transcription by an RNA polymerase, and are able to position small molecules and protein components with nanoscale precision.

Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colours. Structural colours consist of DNA structures, protein labels, native RNA structures or a combination of all three. The sequence of structural colours of RNA identifiers enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) method reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one-step, enzyme-free reaction in a complex human transcriptome using single-molecule read-out. A method has been developed to identify RNA transcript isoforms at the single-molecule level using solid-state nanopore microscopy. In this method, target RNA is refolded into RNA identifiers with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colours that enables identification and quantification using solid-state nanopore microscopy.

Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology. FRET has been used to study protein conformational changes but has never been applied to RNA aptamers. Here the authors develop a genetically encodable RNA aptamer-based FRET system on single-stranded RNA origami scaffolds, and demonstrate it can be used to study RNA conformational changes.

Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.