Injectable Micro-Hydrogel for DNA Delivery: A Promising Therapeutic Platform.

Utilizing the immune system as a strategy for disease prevention and treatment is promising, especially with dendritic cells (DCs) playing a central role in adaptive immune responses. The unique properties of DCs drive interest in developing materials for cell-based therapy and immune modulation. Injectable systems require syringe-compatible scaffolds, while hydrogels, like alginate, known for their programmability and biocompatibility, offer a versatile platform for immune medicine enhancement through easy preparation and room-temperature cross-linking. In this study, we synthesized alginate balls loaded with DCs or cytosine-phosphorothioate-guanine deoxyribonucleotide (CpG DNA) microparticles, aiming for long-term immune cell culture with potential immune stimulation effects. Encapsulated DCs exhibited proliferation within the alginate balls for up to 7 days, and CpG MPs were uniformly dispersed, which can facilitate uptake by DCs. This was supported by the result that DCs effectively phagocytosed CpG microparticles in a 2D environment. After the uptake of CpG MPs, the alginate balls with CpG-MP-uptaken DCs were synthesized successfully. The injectable properties of the alginate balls were easily modulated by adjusting the syringe needle gauges. This innovative strategy holds substantial promise for advancing medical treatments, offering effective and comfortable solutions for controlled immune modulation.

Multimodal Golden DNA Superstructures (GDSs) for Highly Efficient Photothermal Immunotherapy.

DNA-templated metallization has emerged as an efficient strategy for creating nanoscale-metal DNA hybrid structures with a desirable conformation and function. Despite the potential of DNA-metal hybrids, their use as combinatory therapeutic agents has rarely been examined. Herein, we present a simple approach for fabricating a multipurpose DNA superstructure that serves as an efficient photoimmunotherapy agent. Specifically, we adsorb and locally concentrate Au ions onto DNA superstructures through induced local reduction, resulting in the formation of Au nanoclusters. The mechanical and optical properties of these metallic nanoclusters can be rationally controlled by their conformations and metal ions. The resulting golden DNA superstructures (GDSs) exhibit significant photothermal effects that induce cancer cell apoptosis. When sequence-specific immunostimulatory effects of DNA are combined, GDSs provide a synergistic effect to eradicate cancer and inhibit metastasis, demonstrating potential as a combinatory therapeutic agent for tumor treatment. Altogether, the DNA superstructure-templated metal casting system offers promising materials for future biomedical applications.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease.

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dose-dependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Self-assembly of a multimeric genomic hydrogel via multi-primed chain reaction of dual single-stranded circular plasmids for cell-free protein production.

Recent technical advances in cell-free protein synthesis (CFPS) offer several advantages over cell-based expression systems, including the application of cellular machinery, such as transcription and translation, in the test tube. Inspired by the advantages of CFPS, we have fabricated a multimeric genomic DNA hydrogel (mGD-gel) via rolling circle chain amplification (RCCA) using dual single-stranded circular plasmids with multiple primers. The mGD-gel exhibited significantly enhanced protein yield. In addition, mGD-gel can be reused at least five times, and the shape of the mGD-gel can be easily manipulated without losing the feasibility of protein expression. The mGD-gel platform based on the self-assembly of multimeric genomic DNA strands (mGD strands) has the potential to be used in CFPS systems for a variety of biotechnological applications.

Multimeric RNAs for efficient RNA-based therapeutics and vaccines.

There has been a growing interest in RNA therapeutics globally, and much progress has been made in this area, which has been further accelerated by the clinical applications of RNA-based vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Following these successful clinical trials, various technologies have been developed to improve the efficacy of RNA-based drugs. Multimerization of RNA therapeutics is one of the most attractive approaches to ensure high stability, high efficacy, and prolonged action of RNA-based drugs. In this review, we offer an overview of the representative approaches for generating repetitive functional RNAs by chemical conjugation, structural self-assembly, enzymatic elongation, and self-amplification. The therapeutic and vaccine applications of engineered multimeric RNAs in various diseases have also been summarized. By outlining the current status of multimeric RNAs, the potential of multimeric RNA as a promising treatment strategy is highlighted.

DNA Optoelectronics: Versatile Systems for On-Demand Functional Electrochemical Applications.

Herein, we propose innovative deoxyribonucleic acid (DNA)-based gels and their applications in diverse optoelectronics. We prepared the optoelectronic DNA-based gels (OpDNA Gel) through molecular complexation, that is, groove binding and ionic interactions of DNA and 1,1'-diheptyl-4,4'-bipyridinium (DHV). This process is feasible even with sequence-nonspecific DNA extracted from nature (e.g., salmon testes), resulting in the expansion of the application scope of DNA-based gels. OpDNA Gel possessed good mechanical characteristics (e.g., high compressibility, thermoplasticity, and outstanding viscoelastic properties) that have not been observed in typical DNA hydrogels. Moreover, the electrochromic (EC) characteristics of DHV were not lost when combined with OpDNA Gel. By taking advantage of the facile moldability, voltage-tunable EC behavior, and biocompatibility/biodegradability of OpDNA Gel, we successfully demonstrated its applicability in a variety of functional electrochemical systems, including on-demand information coding systems, user-customized EC displays, and microorganism monitoring systems. The OpDNA Gel is a promising platform for the application of DNA-based biomaterials in electrochemical optoelectronics.

RNA polymerization actuating nucleic acid membrane (RANAM)-based biosensing for universal RNA virus detection.

The coronavirus disease (COVID-19) pandemic has shown the importance of early disease diagnosis in preventing further infection and mortality. Despite major advances in the development of highly precise and rapid detection approaches, the time-consuming process of designing a virus-specific diagnostic kit has been a limiting factor in the early management of the pandemic. Here, we propose an RNA polymerase activity-sensing strategy utilizing an RNA polymerization actuating nucleic acid membrane (RANAM) partially metallized with gold for colorimetric RNA virus detection. Following RANAM-templated amplification of newly synthesized RNA, the presence of the RNA polymerase was determined by visualization of the inhibition of an oxidation/reduction (redox) reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and blocked Au3+. As a proof of concept, a viral RNA-dependent RNA polymerase (RdRP), which is found in various RNA virus-infected cells, was chosen as a target molecule. With this novel RANAM biosensor, as little as 10 min of RdRP incubation could significantly reduce the colorimetric signal. Further development into an easy-to-use prototype kit in viral infection diagnosis detected RdRP present at levels even as low as 100 aM. Color formation based on the presence of RdRP could be simply and clearly confirmed through smartphone-assisted color imaging of the prototype kit. This study provides a non-PCR-based RNA virus detection including its variants using RdRP-mediated polymerization.

Construction of a two-dimensional DNA-RNA hybridized membrane for collecting tumor-derived exosomes.

Macroscopic nucleic acid-based structures have attracted much attention in biomedical fields. Here, we introduce a novel DNA-RNA hybridized membrane structure via enzymatic dual polymerization. The membrane exhibited enhanced rigidity and functionality. Encoded with an aptamer, the membrane showed great potential as a collecting platform of tumor-derived exosomes without additional labeling.

Viscosity-Regulated Control of RNA Microstructure Fabrication.

The development of RNA self-assemblies offers a powerful platform for a wide range of biomedical applications. The fabrication process has become more elaborate in order to achieve functional structures with maximized potential. As a facile means to control the structure, here, we report a new approach to manipulate the polymerization rate and subsequent self-assembly process through regulation of the reaction viscosity. As the RNA polymerization rate has a dependence on solution viscosity, the resulting assembly, crystallization, and overall sizes of the product could be manipulated. The simple and precise control of RNA polymerization and self-assembly by reaction viscosity will provide a way to widen the utility of RNA-based materials.

Astrocyte-derived extracellular vesicles enhance the survival and electrophysiological function of human cortical neurons in vitro.

Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies.

Rapid Diagnosis of Coronavirus by RNA-Directed RNA Transcription Using an Engineered RNA-based Platform.

A coronavirus disease (COVID-19) outbreak associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading widely through person-to-person transmission. Various detection approaches have been developed involving quantitative polymerase chain reaction (qPCR) methods, CRISPR-based systems, and direct targeting of specific coronavirus proteins. However, there have only been a few reports on the detection of RNA-dependent RNA polymerase (RdRP), the primer-independent RNA-replicable protein produced by the RNA genes of coronavirus. Here, we introduce a novel diagnostic methodology for COVID-19 using the RNA-directed and de novo RNA replicable function of RdRP. We devised an RNA platform for RdRP-induced transcription (RPRIT) that includes an RNA template that can be directly transcribed by RdRP. By utilizing RPRIT, the presence of RdRP can be readily confirmed within 30 min using isothermal incubation without PCR. This RdRP detection method can provide a new route for rapid diagnosis of RNA virus-infected patients.

Double Controlled Release of Therapeutic RNA Modules through Injectable DNA-RNA Hybrid Hydrogel.

Advances in the DNA nanotechnology have enabled the fabrication of DNA-based hydrogels with precisely controlled structures and tunable mechanical and biological properties. Compared to DNA hydrogel, preparation of RNA-based hydrogel remains challenging due to the inherent instability of naked RNA. To overcome these limitations, we fabricated a DNA-RNA hybrid hydrogel via stepwise dual enzymatic polymerization. Multimeric short hairpin RNAs (shRNAs) were hybridized with functional DNA aptamers for targeting and mechanical properties of the hydrogel. The obtained DNA-RNA hybrid hydrogel was ultrasoft, robust, and injectable hence reconfigurable into any confined structures. As a model system, the hydrogel was able to mimic microtubule structures under physiological conditions and designed to release the functional small interfering RNA (siRNA)-aptamer complex (SAC) sequentially. In addition, we encoded restriction enzyme-responsive sites in DNA-RNA hybrid hydrogel to boost the release of SAC. This novel strategy provides an excellent platform for systematic RNA delivery through double-controlled release, SAC release from hydrogel, and subsequent release of siRNA from the SAC, which has promising potential in RNA therapy.

An enzymatically self-assembled DNA patch for enhanced blood coagulation.

DNA has been widely investigated as a carrier for drug delivery. Here, we describe a macroscopic DNA film that has been generated enzymatically. This DNA film was subsequently coated with thrombin (TB) using an aptamer-protein interaction, to expedite hemostasis over a large area. The DNA film coated with TB (DNA patch) significantly improved plasma hemostasis by acting as a coagulative scaffold for TB, as well as carrying localized TB. This study elucidates the benefits of using enzymatic amplification-based DNA structures in the context of topical drug treatment.

Focused ultrasound-triggered chemo-gene therapy with multifunctional nanocomplex for enhancing therapeutic efficacy.

Nanotechnology-based combination therapies, especially chemo-gene therapy, have been spotlighted as promising alternatives for cancer therapy. However, only a small amount of systemically administered nanomedicines reach the tumor site by the enhanced permeability and retention (EPR) effect, resulting in the limited therapeutic efficacy. Furthermore, the design of ideal drug delivery system for chemo-gene therapy has been impeded by the chemical and physical differences between nucleic acids and chemotherapeutics. Herein, we report a precisely designed nanocomplex which exhibits a focused ultrasound (FU)-responsive release and enhanced accumulation of released therapeutics to tumor site. After the nanocomplex composed of siRNA nanoparticles (siRNA-NP) and chemotherapeutics-loaded microbubbles was systemically injected, the nanocomplex was collapsed around the tumor tissue by FU exposure, and both siRNA-NP and chemotherapeutics were penetrated the dense extracellular matrix (ECM) of tumor site, leading to the enhanced chemo-gene therapeutic efficacy. The two-in-one nanocomplex is expected as a promising platform for combination therapy that can enhance the therapeutic efficiency of combination drugs at the cell and/or tissue levels with high drug loading ratio.

Engineered extracellular vesicles and their mimetics for clinical translation.

Cells secrete extracellular vesicles (EVs) to external environments to achieve cellular homeostasis and cell-to-cell communication. Their therapeutic potential has been constantly spotlighted since they mirror both cytoplasmic and membranous components of parental cells. Meanwhile, growing evidence suggests that EV engineering could further promote EVs with a maximized capacity. In this review, a range of engineering techniques as well as upscaling approaches to exploit EVs and their mimetics are introduced. By laying out the pros and cons of each technique from different perspectives, we sought to provide an overview potentially helpful for understanding the current state of the art EV engineering and a guideline for choosing a suitable technique for engineering EVs. Furthermore, we envision that the advances in each technique will give rise to the combinatorial engineering of EVs, taking us a step closer to a clinical translation of EV-based therapeutics.

BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo.

USE1 has been demonstrated to play crucial roles in the development and progression of human lung cancer. However, the antitumor efficacy of RNA interference (RNAi) targeting of USE1 has not yet been evaluated as a possible clinical application. We here synthesized USE1 targeting bubbled RNA-based cargo (BRC) composed of densely packed multimeric pre-siRNAs with specific Dicer cleavage sites to enable efficient siRNA release upon entry to target cells. The physical entanglement and continuous networking of RNAs via hybridization during enzymatic replication serve as a driving force for the self-assembly of BRCs. These molecules effectively suppressed the transcription of their target genes, leading to tumor growth suppression in vitro and in vivo. Moreover, their repeated intravenous administration efficiently inhibited the growth of A549 tumor xenografts. Based on these findings of a reduced cancer cell viability following a USE1 knockdown, we further explored cell cycle arrest and apoptosis pathways. The observed tumor cell growth suppression was found to be controlled by cell cycle arrest and apoptosis signals induced by the USE1 reduction. These results suggest that USE1 BRCs may have future clinical applications as an RNAi-based cancer therapy.

Sustained Release of Minor-Groove-Binding Antibiotic Netropsin from Calcium-Coated Groove-Rich DNA Particles.

Control of the release properties of drugs has been considered a key factor in the development of drug delivery systems (DDSs). However, drug delivery has limitations including cytotoxicity, low loading efficiency, and burst release. To overcome these challenges, nano or micro-particles have been suggested as carrier systems to deliver chemical drugs. Herein, nano-sized DNA particles (DNAp) were manufactured to deliver netropsin, which is known to bind to DNA minor grooves. The rationally designed particles with exposed rich minor grooves were prepared by DNAp synthesis via rolling circle amplification (RCA). DNAp could load large quantities of netropsin in its minor grooves. An analytical method was also developed for the quantification of netropsin binding to DNAp by UV-visible spectrometry. Moreover, controlled release of netropsin was achieved by forming a layer of Ca2+ on the DNAp (CaDNAp). As a proof of concept, the sustained release of netropsin by CaDNAp highlights the potential of the DNAp-based delivery approach.

Self-assembled DNA hollow spheres from microsponges.

We report a novel approach for generating nanosized DNA hollow spheres (HSs) using enzymatically produced DNA microsponges in a self-templating manner. In previous studies, preparation of DNA nanostructures with specified functions required multiple complicated steps. In this study, however, a simple treatment with the nucleophilic agent 4-dimethylaminopyridine (DMAP) enabled a gradual disentanglement of DNA in microsponges by electrostatic interactions between DMAP and DNA, and the DNA underwent a reassembly process to generate hollow shell structures without denaturation/annealing by thermal cycling. In addition, this synthetic process was conducted in a water-based system without organic solvents, enabling the synthesis of biologically and environmentally friendly products. Based on the benefits of hollow shell structures, which include their high surface-to-volume ratio and ability to encapsulate small molecules, we envision that this simple approach for synthesizing DNA HSs will provide a new platform for maximizing their potential use in drug delivery and bio-imaging.

Immunostimulatory Effects Triggered by Self-Assembled Microspheres with Tandem Repeats of Polymerized RNA Strands.

Self-assembled RNA particles have been exploited widely to maximize the therapeutic potential of RNA. However, the immune response via RNA particles is not fully understood. In addition, the investigation of the immunogenicity from RNA-based particles is required owing to inherent immunostimulatory effects of RNA for clinical translation. To examine the immune stimulating potency, rationally designed microsized RNA particles, called RNA microspheres (RMSs), are generated with single or double strands via rolling circle transcription. The RMSs show an exceptional stability in the presence of serum, while they are selectively degraded under endolysosomal conditions. With precisely controlled size, both RMSs are successfully taken up by macrophages. Unlike the nature of RNA fragments, RMSs induce only basal-level expression of inflammatory cytokines as well as type I interferon from macrophages, suggesting that RMSs are immunocompatible in the therapeutic dose range. Taken together, this study could help accelerate clinical translation and broaden the applicability of the self-assembled RNA-based particles without being limited by their potential immunotoxicity, while a systematic controllability study observing the release of RNA fragments from RMSs would provide self-assembled RNA-based structures with a great potential for immunomodulation.

Correction: CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells.

[This corrects the article DOI: 10.1039/C7RA13293J.].