Nucleic Acid Delivery Systems Research Articles

A new class of polyphenolic carbosilane dendrimers binds human serum albumin in a structure-dependent fashion

The use of dendrimers as drug and nucleic acid delivery systems requires knowledge of their interactions with objects on their way to the target. In the present work, we investigated the interaction of a new class of carbosilane dendrimers functionalized with polyphenolic and caffeic acid residues with human serum albumin, which is the most abundant blood protein. The addition of dendrimers to albumin solution decreased the zeta potential of albumin/dendrimer complexes as compared to free albumin, increased density of the fibrillary form of albumin, shifted fluorescence spectrum towards longer wavelengths, induced quenching of tryptophan fluorescence, and decreased ellipticity of circular dichroism resulting from a reduction in the albumin α-helix for random coil structural form. Isothermal titration calorimetry showed that, on average, one molecule of albumin was bound by 6–10 molecules of dendrimers. The zeta size confirmed the binding of the dendrimers to albumin. The interaction of dendrimers and albumin depended on the number of caffeic acid residues and polyethylene glycol modifications in the dendrimer structure. In conclusion, carbosilane polyphenolic dendrimers interact with human albumin changing its structure and electrical properties. However, the consequences of such interaction for the efficacy and side effects of these dendrimers as drug/nucleic acid delivery system requires further research.

Fluorinated Lipid Nanoparticles for Enhancing mRNA Delivery Efficiency.

Lipid nanoparticles (LNPs), a nonviral nucleic acid delivery system, have shown vast potential for vaccine development and disease treatment. LNPs assist mRNA to cross physiological barriers such as cell membranes and endosomes/lysosomes, promoting the intracellular presentation of mRNA. However, the endosome escape efficiency and biosafety of currently commercialized LNPs are still unsatisfactory, resulting in underutilization of mRNA. Herein, we report that fluorinated modification of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (PEG-DSPE), termed as FPD, in the LNPs can improve the delivery efficiency of mRNA. FPD accounts for only 1.5% of lipids in LNPs but could mediate a 5-fold and nearly 2-fold enhancement of mRNA expression efficiency in B16F10 tumor cells and primary dendritic cells, respectively. Mechanism studies reveal that FPD promotes the cellular internalization of LNPs as well as endosome escape. In vivo studies substantiate that FPD can augment overall mRNA expression at least 3-fold, either by intravenous or intraperitoneal injection, compared to LNPs prepared with nonfluorinated PEG-lipids at a relatively low mRNA dose. Besides, with the introduction of FPD, mRNA expression in the spleen augmented compared to that of the DMG-PEG commercial formulations. Benefiting from a prudent dosage of fluorine, the fluorinated LNPs display favorable biosafety profiles at cellular and zoological levels.

One Endothelium-Targeted Combined Nucleic Acid Delivery System for Myocardial Infarction Therapy.

Acute myocardial infarction (MI) and ischemic heart disease are the leading causes of heart failure and mortality. Currently, research on MI treatment is focused on angiogenic and anti-inflammatory therapies. Although endothelial cells (ECs) are critical for triggering inflammation and angiogenesis, no approach has targeted them for the treatment of MI. In this study, we proposed a nonviral combined nucleic acid delivery system consisting of an EC-specific polycation (CRPPR-grafted ethanolamine-modified poly(glycidyl methacrylate), CPC) that can efficiently codeliver siR-ICAM1 and pCXCL12 for the treatment of MI. Animals treated with the combination therapy exhibited better cardiac function than those treated with each nucleic acid alone. In particular, the combination therapy of CPC/siR-ICAM1 and CPC/pCXCL12 significantly improved cardiac systolic function, anti-inflammatory responses, and angiogenesis compared to the control group. In conclusion, CPC-based combined gene delivery systems show impressive performance in the treatment of MI and provide a programmed strategy for the development of codelivery systems for various EC-related diseases.

Comparative analysis of nucleic acid delivery systems for gene therapy: assessing viral and non-viral approaches with emphasis on extracellular vesicles

Comparative analysis of nucleic acid delivery systems for gene therapy: assessing viral and non-viral approaches with emphasis on extracellular vesicles

Cell-Penetrating Peptides as Keys to Endosomal Escape and Intracellular Trafficking in Nanomedicine Delivery.

This review article discusses the challenges of delivering cargoes to the cytoplasm, for example, proteins, peptides, and nucleic acids, and the mechanisms involved in endosomal escape. Endocytosis, endosomal maturation, and exocytosis pose significant barriers to effective cytoplasmic delivery. The article explores various endosomal escape mechanisms, such as the proton sponge effect, osmotic lysis, membrane fusion, pore formation, membrane destabilization/ disruption, and vesicle budding and collapse. Additionally, it discusses the role of lysosomes, glycocalyx, and molecular crowding in the cytoplasmic delivery process. Despite the recent advances in nonviral delivery systems, there is still a need to improve cytoplasmic delivery. Strategies such as fusogenic peptides, endosomolytic polymers, and cell-penetrating peptides have shown promise in improving endosomal escape and cytoplasmic delivery. More research is needed to refine these strategies and make them safer and more effective. In conclusion, the article highlights the challenges associated with cytoplasmic delivery and the importance of understanding the mechanisms involved in endosomal escape. A better understanding of these processes could result in the creation of greater effectiveness and safe delivery systems for various cargoes, including proteins, peptides, and nucleic acids.

Nanoparticles derived from herbal preparations May represent a novel nucleic acid therapy

AbstractA significant number of nucleic acid therapies have been approved, highlighting the vast potential of nucleic acid therapeutics. Although numerous delivery strategies have been investigated, and progress has been made in nucleic acid delivery systems, the oral delivery route still presents a significant obstacle. Recently, a novel and promising force in oral nucleic acid delivery has emerged, namely Decoctosomes and Bencaosomes, obtained from the decoction of natural herbs, which refers to boiled Chinese herbal medicines. The active components of Decoctosomes include small molecules, peptides, and small RNAs (sRNAs). sRNAs were the first targets of extensive research. Additionally, Bencaosomes have been proven effective in several disease mouse models as artificially enhanced decoctosome products. This perspective summarizes the development and possible future directions of Decoctosomes and Bencaosomes, two varieties of nanoparticles that may open the door to new possibilities in nucleic acid therapy.

Effect of the size of nucleic acid delivery systems on their fate in cancer treatment

Nucleic acid therapeutics are emerging as a promising class of medicines, offering unique therapeutic options for cancer at the gene level. However, the druggability of nucleic acid therapeutics is fundamentally restricted by their low stability, poor membrane permeability, and low bioavailability, necessitating the use of delivery vectors. Various delivery vectors have been developed for nucleic acid therapeutics. The fate of established nucleic acid delivery systems (NADS) in vivo substantially affects the delivery efficiency and therapeutic efficacy. The physicochemical properties of NADS (such as size, charge, shape, etc) are crucial for the interaction of NADS with various biological barriers in the body, thereby determining the fate of NADS in the body. Nanoparticle (NP) size is an important parameter defining the blood circulation, distribution, tumor accumulation, and cellular uptake of NADS. This mini-review briefly introduces the various biological barriers of NADS in cancer treatment and focuses on the influence of the particle size of delivery vectors on the in vivo fate of NADS and their therapeutic efficacy, which provides new insights into the rational design of NADS.

Induced Self-Assembly of Vitamin E-Spermine/siRNA Nanocomplexes via Spermine/Helix Groove-Specific Interaction for Efficient siRNA Delivery and Antitumor Therapy.

Gene therapy has been one of potential strategies for the treatment of different diseases, where efficient and safe gene delivery systems are also extremely in need. Current lipid nanoparticles (LNP) technology highly depends on the packing and condensation of nucleic acids with amine moieties. Here, an attempt to covalently link two natural compounds, spermine and vitamin E, is made to develop self-assembled nucleic acid delivery systems. Among them, the spermine moieties specifically interact with the major groove of siRNA helix through salt bridge interaction, while vitamin E moieties are located around siRNA duplex. Such amphiphilic vitamin E-spermine/siRNA complexes can further self-assemble into nanocomplexes like multiblade wheels. Further studies indicate that these siRNA nanocomplexes with the neutrally charged surface of vitamin E can enter cells via caveolin/lipid raft mediated endocytosis pathway and bypass lysosome trapping. With these self-assembled delivery systems, efficient siRNA delivery is successfully achieved for Eg5 and Survivin gene silencing as well as DNA plasmid delivery. Further in vivo study indicates that VE-Su-Sper/DSPE-PEG2000/siSurvivin self-assembled nanocomplexes can accumulate in cancer cells and gradually release siRNA in tumor tissues and show significant antitumor effect in vivo. The self-assembled delivery system provides a novel strategy for highly efficient siRNA delivery.

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Selection of GalNAc-Conjugated siKeap1 as Disease-Specific Delivery System for Chemotherapy-Induced Liver Injury and Chronic Liver Disease.

Chemotherapy-induced liver injury (CILI) is a pressing concern in cancer patients. One promising approach involves activating nuclear factor erythroid 2-related factor 2 (Nrf2) to mitigate CILI. However, selectively activating liver Nrf2 without compromising chemotherapy's efficacy has remained elusive. Herein, two RNAi delivery strategies were explored: lipid nanoparticle (LNP) and N-acetylgalactosamine (GalNAc) delivery systems loaded with siRNA designed to silence Kelch-like-ECH associated protein 1 (Keap1) by aiming for liver-specific Nrf2 activation. Remarkably, siKeap1-LNP exhibited unintended tumor targeting alongside liver effects, thereby potentially promoting tumor progression. Conversely, siKeap1-GalNAc did not compromise chemotherapy efficacy and outperformed the conventional Nrf2 activator, bardoxolone, in mitigating CILI. This study proposes siKeap1-GalNAc as a promising therapeutic avenue for liver injury. Importantly, our study bridges a crucial gap concerning the delivery system for liver targeting but not tumor targeting and underscores the importance of selecting nucleic acid delivery systems tailored to specific diseases, not just to specific organs.

Characterization of N-glycosylation and its functional role in SIDT1-Mediated RNA uptake

The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multi-pass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we employ high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system, with potential implications for therapeutic applications.

Tracking the Endosomal Escape of Nanoparticles in Live Cells Using a Triplex‐Forming Oligonucleotide

AbstractNanoparticle‐mediated intracellular delivery of oligonucleotides is a complex phenomenon that depends on the architecture and the intracellular trafficking of the engineered nanoparticles. Unravelling the molecular arrangements of oligonucleotides within the nanoparticles as well as their intracellular behavior are essential for designing effective nucleic acid delivery systems. Herein, a simple and general strategy for probing the endosomal escape of nanoparticles carrying oligonucleotides in live cells is reported. A triplex‐forming oligonucleotide probe is designed to target the transcription factor, kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), in the cytosol of cells and to transduce the binding into a fluorescent Förster resonance energy transfer (FRET) signal. The combined use of the triplex‐forming oligonucleotide probe and super‐resolution microscopy enables the elucidation of the morphology, intracellular localization, and endosomal escape of the oligonucleotide‐loaded nanoparticles on a molecular level and with nanoscale resolution. The co‐delivery of the FRET probe and mRNA in cells via lipid‐ and polymer‐ based nanoparticles allow simultaneous correlation of the endosomal escape properties of nanoparticles and gene expression efficiency.

DNA nanotechnology-based nucleic acid delivery systems for bioimaging and disease treatment.

Nucleic acids, including DNA and RNA, have been considered as powerful and functional biomaterials owing to their programmable structure, good biocompatibility, and ease of synthesis. However, traditional nucleic acid-based probes have always suffered from inherent limitations, including restricted cell internalization efficiency and structural instability. In recent years, DNA nanotechnology has shown great promise for the applications of bioimaging and drug delivery. The attractive superiorities of DNA nanostructures, such as precise geometries, spatial addressability, and improved biostability, have enabled them to be a novel category of nucleic acid delivery systems for biomedical applications. In this review, we introduce the development of DNA nanotechnology, and highlight recent advances of DNA nanostructure-based delivery systems for cellular imaging and therapeutic applications. Finally, we propose the challenges as well as opportunities for the future development of DNA nanotechnology in biomedical research.

Unveiling Sticholysin II and plasmid DNA interaction: Implications for developing non-viral vectors

Non-viral gene delivery systems offer significant potential for gene therapy due to their versatility, safety, and cost advantages over viral vectors. However, their effectiveness can be hindered by the challenge of efficiently releasing the genetic cargo from endosomes to prevent degradation in lysosomes. To overcome this obstacle, functional components can be incorporated into these systems. Sticholysin II (StII) is one of the pore-forming proteins derived from the sea anemone Stichodactyla helianthus, known for its high ability to permeabilize cellular and model membranes. In this study, we aimed to investigate the interaction between StII, and a model plasmid (pDNA) as an initial step towards designing an improved vector with enhanced endosomal escape capability. The electrophoretic mobility shift assay (EMSA) confirmed the formation of complexes between StII and pDNA. Computational predictions identified specific residues involved in the StII-DNA interaction interface, highlighting the importance of electrostatic interactions and hydrogen bonds in mediating the binding. Atomic force microscopy (AFM) of StII-pDNA complexes revealed the presence of nodular fiber and toroid shapes. These complexes were found to have a predominantly micrometer size, as confirmed by dynamic light scattering (DLS) measurements. Despite increase in the overall charge, the complexes formed at the evaluated nitrogen-to-phosphorus (N/P) ratios still maintained a negative charge. Moreover, StII retained its pore-forming capacity regardless of its binding to the complexes. These findings suggest that the potential ability of StII to permeabilize endosomal membranes could be largely maintained when combined with nucleic acid delivery systems. Additionally, the still remaining negative charge of the complexes would enable the association of another positively charged component to compact pDNA. However, to minimize non-specific cytotoxic effects, it is advisable to explore methods to regulate the protein's activity in response to the microenvironment.

Computational approaches to lipid-based nucleic acid delivery systems

Nucleic acid-based therapies have shown enormous effectiveness as vaccines against the recent COVID19 pandemics and hold great promises in the fight of a broad spectrum of diseases ranging from viral infections to cancer up to genetically transmitted pathologies. Due to their highly degradable polyanionic nature, nucleic acids need to be packed in sophisticate delivery vehicles which compact them up, protect them from early degradation and help delivery them to the right tissue/cells. Lipid-based nanoparticles (LNP) represent, at present, the main solution for nucleic acid delivery. They are made of a mixture of lipids whose key ingredient is an ionizable cationic lipid. Indeed, the interactions between the polyanionic nucleic acids and the ionizable cationic lipids, and their pH-dependent regulation in the life cycle of the nanoparticle, from production to cargo delivery, mostly determine the effectiveness of the therapeutic approach. Notwithstanding the large improvements in the delivery efficiency of LNPs in the last two decades, it is estimated that only a small fraction of the cargo is actually delivered, stimulating further research for the design of more effective LNP formulations. A rationally driven design would profit from the knowledge of the precise molecular structure of these materials, which is however still either missing or characterized by poor spatial resolution. Computational approaches have often been used as a molecular microscope either to enrich the available experimental data and provide a molecular-level picture of the LNPs or even simulate specific processes involving the formation and/or the molecular mechanisms of action of the LNP. Here, I review the recent literature in the field.

Mechanistic Insights into Alternative Gene Splicing in Oxidative Stress and Tissue Injury.

Significance: Oxidative stress (OS) and inflammation are inducers of tissue injury. Alternative splicing (AS) is an essential regulatory step for diversifying the eukaryotic proteome. Human diseases link AS to OS; however, the underlying mechanisms must be better understood. Recent Advances: Genome‑wide profiling studies identify new differentially expressed genes induced by OS-dependent ischemia/reperfusion injury. Overexpression of RNA-binding protein RBFOX1 protects against inflammation. Hypoxia-inducible factor-1α directs polypyrimidine tract binding protein 1 to regulate mouse carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) AS under OS conditions. Heterogeneous nuclear ribonucleoprotein L variant 1 contains an RGG/RG motif that coordinates with transcription factors to influence human CEACAM1 AS. Hypoxia intervention involving short interfering RNAs directed to long-noncoding RNA 260 polarizes M2 macrophages toward an anti-inflammatory phenotype and alleviates OS by inhibiting IL-28RA gene AS. Critical Issues: Protective mechanisms that eliminate reactive oxygen species (ROS) are important for resolving imbalances that lead to chronic inflammation. Defects in AS can cause ROS generation, cell death regulation, and the activation of innate and adaptive immune factors. We propose that AS pathways link redox regulation to the activation or suppression of the inflammatory response during cellular stress. Future Directions: Emergent studies using molecule-mediated RNA splicing are being conducted to exploit the immunogenicity of AS protein products. Deciphering the mechanisms that connect misspliced OS and pathologies should remain a priority. Controlled release of RNA directly into cells with clinical applications is needed as the demand for innovative nucleic acid delivery systems continues to be demonstrated.

Engineered Silica Nanoparticles for Nucleic Acid Delivery.

The development of nucleic acid-based drugs holds great promise for therapeutic applications, but their effective delivery into cells is hindered by poor cellular membrane permeability and inherent instability. To overcome these challenges, delivery vehicles are required to protect and deliver nucleic acids efficiently. Silica nanoparticles (SiNPs) have emerged as promising nanovectors and recently bioregulators for gene delivery due to their unique advantages. In this review, a summary of recent advancements in the design of SiNPs for nucleic acid delivery and their applications is provided, mainly according to the specific type of nucleic acids. First, the structural characteristics and working mechanisms of various types of nucleic acids are introduced and classified according to their functions. Subsequently, for each nucleic acid type, the use of SiNPs for enhancing delivery performance and their biomedical applications are summarized. The tailored design of SiNPs for selected type of nucleic acid delivery will be highlighted considering the characteristics of nucleic acids. Lastly, the limitations in current research and personal perspectives on future directions in this field are presented. It is expected this opportune review will provide insights into a burgeoning research area for the development of next-generation SiNP-based nucleic acid delivery systems.

Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids.

Recent developments in artificial nucleic acid and drug delivery systems present possibilities for the symbiotic engineering of therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs) and small interfering ribonucleic acids (siRNAs). Employing these technologies, triplex-forming oligonucleotides (TFOs) or peptide nucleic acids (PNAs) can be applied to the development of symbiotic genome-targeting tools as well as a new class of oligonucleotide drugs, which offer conceptual advantages over antisense as the antigene target generally comprises two gene copies per cell rather than multiple copies of mRNA that are being continually transcribed. Further, genome editing by TFOs or PNAs induces permanent changes in the pathological genes, thus facilitating the complete cure of diseases. Nuclease-based gene-editing tools, such as zinc fingers, CRISPR-Cas9, and TALENs, are being explored for therapeutic applications, although their potential off-target, cytotoxic, and/or immunogenic effects may hinder their in vivo applications. Therefore, this review is aimed at describing the ongoing progress in TFO and PNA technologies, which can be symbiotic genome-targeting tools that will cause a near-future paradigm shift in drug development.

Quality by Design (QbD) and Design of Experiments (DOE) as a Strategy for Tuning Lipid Nanoparticle Formulations for RNA Delivery.

The successful development of nonviral delivery systems for nucleic acids has been reported extensively over the past years. Increasingly employed to improve the delivery efficiency and therapeutic efficacy of RNA are lipid nanoparticles (LNPs). Many of the various critical formulation parameters can affect the quality attributes and effectiveness of these nano-formulations. Therefore, the systematic drug development approach (QbD) and multivariate design and statistical analysis (DOE) can be very helpful and recommended for the optimization of the composition and production of RNA-LNPs. This review addresses the concepts and applications of QbD and/or DOE for the development of lipid nanoparticles for the delivery of different types of RNA, reporting examples published in the ten recent years presenting the latest trends and regulatory requirements as well as the modern mathematical and statistical design methods. As the topic explored in this review is a novel approach, the full QbD has been described in only a few papers, and a few refer only to some aspects of QbD. In contrast, the DOE approach has been used in most of the optimization works. Different approaches and innovations in DOE have been observed. Traditional statistical tests and modeling (ANOVA, regression analysis) are slowly being replaced by artificial intelligence and machine learning methods.

Engineering and Delivery of cGAS-STING Immunomodulators for the Immunotherapy of Cancer and Autoimmune Diseases.

The cyclic GMP-AMP synthase-stimulator interferon gene (cGAS-STING) pathway is an emerging therapeutic target for the prophylaxis and therapy of a variety of diseases, ranging from cancer, infectious diseases, to autoimmune disorders. As a cytosolic double stranded DNA (dsDNA) sensor, cGAS can bind with relatively long dsDNA, resulting in conformational change and activation of cGAS. Activated cGAS catalyzes the conversion of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) into cGAMP, a cyclic dinucleotide (CDN). CDNs, including 2'3'-cGAMP, stimulate adapter protein STING on the endoplasmic membrane, triggering interferon regulatory factor 3 (IRF3) phosphorylation and nuclear factor kappa B (NF-κB) activation. This results in antitumor and antiviral type I interferon (IFN-I) responses. Moreover, cGAS-STING overactivation and the resulting IFN-I responses have been associated with a number of inflammatory and autoimmune diseases. This makes cGAS-STING appealing immunomodulatory targets for the prophylaxis and therapy of various related diseases. However, drug development of CDNs and CDN derivatives is challenged by their limited biostability, difficult formulation, poor pharmacokinetics, and inefficient tissue accumulation and cytosolic delivery. Though recent synthetic small molecular CDN- or non-CDN-based STING agonists have been reported with promising preclinical therapeutic efficacy, their therapeutic efficacy and safety remain to be fully evaluated preclinically and clinically. Therefore, it is highly desirable and clinically significant to advance drug development for cGAS-STING activation by innovative approaches, such as drug delivery systems and drug development for pharmacological immunomodulation of cGAS. In this Account, we summarize our recent research in the engineering and delivery of immunostimulatory or immunoregulatory modulators for cGAS and STING for the immunotherapy of cancer and autoimmune diseases. To improve the delivery efficiency of CDNs, we developed ionizable and pH-responsive polymeric nanocarriers to load STING agonists, aiming to improve the cellular uptake and facilitate the endosomal escape to induce efficient STING activation. We also codelivered STING agonists with complementary immunostimulatants in nanoparticle-in-hydrogel composites to synergetically elicit potent innate and adaptive antitumor responses that eradicate local and distant large tumors. Further, taking advantage of the simplicity of manufacturing and the established nucleic acid delivery system, we developed oligonucleotide-based cGAS agonists as immunostimulant immunotherapeutics as well as adjuvants for peptide antigens for cancer immunotherapy. To suppress the overly strong proinflammatory responses associated with cGAS-STING overactivation in some of the autoimmune disorders, we devised nanomedicine-in-hydrogel (NiH) that codelivers a cGAS inhibitor and cell-free DNA (cfDNA)-scavenging cationic nanoparticles (cNPs) for systemic immunosuppression in rheumatoid arthritis (RA) therapy. Lastly, we discussed current drug development by targeting cGAS-STING for cancer, infectious diseases, and autoimmune diseases, as well as the potential opportunities for utilizing cGAS-STING pathway for versatile applications in disease treatment.