Search Results (120)

Background/Objectives: After many years of research and the successful development of therapeutic products by a few industrial actors, the COVID-19 vaccines brought messenger RNAs, as well as other nucleic acid modalities, such as antisense oligonucleotides, small interfering RNA, and aptamers, into the spotlight, eliciting renewed interest from both academia and industry. However, owing to their structure, relative “fragility”, and the (usually) intracellular site of action, the delivery of these therapeutics has frequently proven to be a key limitation, especially when considering endosomal escape, which still needs to be overcome. Methods: By compiling delivery-related data on approved and late clinical-phase ribonucleic acid therapeutics, this review aims to assess the delivery strategies that have proven to be successful or are emerging, as well as areas where more research is needed. Results: In very specific cases, some strategies appeared to be quite effective, such as the N-acetylgalactosamine moiety in the case of liver delivery. Surprisingly, it also appears that for some modalities, efforts in molecular design have led to more “drug-like” properties, enablingthe administration of naked nucleic acids, without any form of encapsulation. This appears to be especially true when local administration, i.e., by injection, is possible, as this provides de facto targeting and a high local concentration, which can compensate for the small proportion of nucleic acids that reach the cytoplasm. Conclusions: Nucleic acid-based therapeutics have come a long way in terms of their physicochemical properties. However, due to their inherent limitations, targeting appears to be crucial for their efficacy, even more so than for traditional pharmaceutical modalities. Full article

Messenger RNA (mRNA) vaccines have exhibited promising potential for infectious disease prevention. Although various delivery methods have been explored, the use of electroporation (EP) for the delivery of naked mRNA has received relatively less attention. In this study, we used mouse models to investigate whether naked mRNA vaccine delivery via intramuscular EP (IM-EP) elicits a protective immune response against lethal viral infection. To achieve this, we injected C57BL/6 mice with naked mRNA encoding the SARS-CoV-2 mRNA vaccine via IM-EP and evaluated the resulting immune responses. IM-EP-mediated delivery of the mRNA vaccine induced robust humoral and cellular immune responses, characterized by elevated SARS-CoV-2 receptor-binding domain (RBD)-specific IgG antibodies, enhanced IFN-γ production by CD8⁺ T cells, and upregulated cytokine expression in the muscle and lymph nodes. Using the K18-hACE2 mouse model, we revealed that IM-EP-mediated delivery of the naked mRNA vaccine effectively protected mice from lethal SARS-CoV-2 infection. Overall, our findings suggest that the delivery of naked mRNA via IM-EP can be an effective strategy for preventing infectious diseases. Full article

RNA interference (RNAi) has emerged as a potent mechanism for combating pathogenic fungi and oomycetes over the past decades. It offers a promising gene-silencing approach by targeting crucial genes involved in diseases caused by economically and scientifically significant fungal pathogens, such as Botrytis cinerea and Fusarium species. Simultaneously, nano-agro-products have gained attention as alternatives to traditional fungicides in plant protection strategies. However, the instability of naked RNA molecules outside the cellular environment presents a challenge, as they degrade rapidly, limiting their efficacy for prolonged disease control. Concerns regarding the toxicity of protective nanoparticles to non-target organisms have also arisen. Integrating RNAi with nano-agro-products, particularly nanocarriers, to form RNA-nano complexes has demonstrated significant potential, providing enhanced RNA stability, reduced toxicity, and extended disease control. This review explores the mechanisms of RNA-nano complexes-mediated plant protection, addressing RNA stability and nano-toxicity issues while examining the prospects of RNA-nano complex research in plant pathogen management. Full article

Transforming growth factor-β₁ (TGF-β₁) promotes the growth and metastasis of lung cancer cells. Therefore, TGF-β₁ siRNA (siTGF-β₁) gene therapy was introduced to inhibit the expression of TGF-β₁ at the nucleic acid level to avert tumor growth and metastasis. However, the delivery of naked siRNA is typically restricted by a short half-life in vivo, difficulties in delivery in vivo, and safety issues. Using siTGF-β₁ as a model drug, we established an actively targeted immunoliposome delivery system to investigate the role of siTGF-β₁ in non-small-cell lung cancer (NSCLC). The results showed that the constructed immune liposomes were in a position to deliver siTGF-β₁ to tumor cells, thus achieving a series of effects such as improving the poor stability and short half-life of naked siRNA. RNA interference of siTGF-β₁ reduced the cell viability, growth, and migration potential of human non-small cell lung cancer cells (A549). Moreover, in an A549 tumor-bearing nude mouse model, siTGF-β₁ transfection markedly reduced tumor growth and tumor volume. Inhibiting TGF-β₁ diminished cancer cell viability and migration and promoted apoptosis in NSCLC, as confirmed by the findings of this study. Therefore, targeting siTGF-β₁ with immunoliposomes may be a new therapeutic strategy for treating non-small-cell lung cancer. Full article

Background/Objectives: Transforming Growth Factor-beta (TGFβ1) plays a core role in the process of pulmonary fibrosis (PF). The progression of pulmonary fibrosis can be alleviated by siRNA-based inhibiting TGF-β1. However, the limitations of naked siRNA lead to the failure of achieving therapeutic effect. This study aimed to design lipid nanoparticles (LNPs) that can deliver siTGF-β1 to the lungs for therapeutic purposes. Methods: The cytotoxicity and transfection assay in vitro were used to screen ionizable lipids (ILs). Design of Experiments (DOE) was used to obtain novel LNPs that can enhance resistance to atomization shear forces. Meanwhile, the impact of LNPs encapsulating siTGF-β1 (siTGFβ1-LNPs) on PF was investigated. Results: When DLin-DMA-MC3 (MC3) was used as the ILs, the lipid phase ratio was MC3:DSPC:DMG-PEG2000:cholesterol = 50:10:3:37, and N/P = 3.25; the siTGFβ1-LNPs could be stably delivered to the lungs via converting the siTGFβ1-LNPs solution into an aerosol (atomization). In vitro experiments have confirmed that siTGFβ1-LNPs have high safety, high encapsulation, and can promote cellular uptake and endosomal escape. In addition, siTGFβ1-LNPs significantly reduced inflammatory infiltration and attenuated deposition of extracellular matrix (ECM) and protected the lung tissue from the toxicity of bleomycin (BLM) without causing systemic toxicity. Conclusions: The siTGFβ1-LNPs can be effectively delivered to the lungs, resulting in the silencing of TGF-β1 mRNA and the inhibition of the epithelial–mesenchymal transition pathway, thereby delaying the process of PF, which provides a new method for the treatment and intervention of PF. Full article

The plant pathogenic fungus Botrytis cinerea causes significant losses in agricultural production and it is rather difficult to control due to its broad host range and environmental persistence. The management of gray mold disease is still mainly based on the use of chemicals, which could have harmful effects not only due to impacts on the environment and human health, but also because they favor the development of fungicide-resistant strains. In this scenario, the strategy of RNA interference (RNAi) is being widely considered, and Spray-Induced Gene Silencing (SIGS) is gaining interest as a versatile, sustainable, effective, and environmentally friendly alternative to the use of chemicals in the protection of crops. The SIGS approach was evaluated to control B. cinerea infection on lettuce plants. In vitro-synthesized dsRNA molecules (BcBmp1-, BcBmp3-, and BcPls1-dsRNAs) were used naked, or complexed to small layered double hydroxide (sLDH) clay nanosheets. Therefore, treatments were applied by pressure spraying whole lettuce plants lately inoculated with B. cinerea. All sprayed dsRNAs proved effective in reducing disease symptoms with a notable reduction compared to controls. The effectiveness of SIGS in reducing disease caused by B. cinerea was high overall and increased significantly with the use of sLDH clay nanosheets. The sLDH clay nanosheet–dsRNA complexes showed better plant protection over time compared to the use of naked dsRNA and this was particularly evident at 27 days post-inoculation. RNAi-based biocontrol could be an excellent alternative to chemical fungicides, and several RNAi-based products are expected to be approved soon, although they will face several challenges before reaching the market. Full article

Background: Although mRNA vaccines encapsulated in lipid nanoparticles (LNPs) have demonstrated a safety profile with minimal serious adverse events in clinical trials, there is opportunity to further reduce mRNA reactogenicity. The development of naked mRNA vaccines could improve vaccine tolerability. Naked nucleic acid delivery using the jet injection method may be a solution. Methods: In the first part of the study, the optimal conditions providing low traumatization and high expression of the model mRNA-GFP molecule in the tissues of laboratory animals were determined. Then, we used the selected protocol to immunize BALB/c mice with mRNA-RBD encoding the SARS-CoV-2 receptor-binding domain (RBD). It was demonstrated that mice vaccinated with naked mRNA-RBD developed a high level of specific antibodies with virus-neutralizing activity. The vaccine also induced a strong RBD-specific T-cell response and reduced the viral load in the lungs of the animals after infection with the SARS-CoV-2 virus. The level of immune response in mice immunized with mRNA-RBD using a spring-loaded jet injector was comparable to that in animals immunized with mRNA-RBD encapsulated in LNPs. Results: In this study, the efficacy of an inexpensive, simple, and safe method of mRNA delivery using a spring-loaded jet injector was evaluated and validated. Conclusions: Our findings suggest that the jet injection method may be a possible alternative to LNPs for delivering mRNA vaccines against SARS-CoV-2 infection. Full article

Among the different naked and quasi-enveloped viruses, the hepatitis A virus (HAV), hepatitis E virus (HEV), and norovirus genogroups I and II (NoV GI and NoV GII) are considered the main microbiological noxae involved in foodborne outbreaks. Mussels can harbor pathogens in their tissues. In addition to epidemiological attention, marine water temperature changes are considered a crucial variable influencing viral loads. This study aimed to biomolecularly screen 1775 farmed mussels (Mytilus galloprovincialis) for viral ribonucleic acid (RNA) sequence detection (belonging to the HAV, HEV, and NoV GI and GII genogroups) in three different sampling periods (spring, summer, and winter), with the mussels collected from three farms located in the Central Adriatic Sea (Italy). The results showed that 10.42% of the screened animals harbored at least one type of pathogen RNA, more specifically, 5.35% HEV, 4.51% NoV GI, and 0.56% HAV. The highest genetic equivalent (GE) amounts were majorly observed in the winter season (NoV GI 1.0 × 10³ GE/g and HEV 1.0 × 10² GE/g), resulting in statistical differences when compared to summer and spring (p-value: <0.001). The original data obtained serve to bring scientific attention to the possible influence of environmental and climatic aspects on viral loads, highlighting the crucial role played by biomolecular assays as preventive medicine tools. Full article

mRNA vaccines were highly effective in response to the COVID-19 pandemic, making them an attractive platform to address cancers and other infectious diseases. Many new mRNA vaccines in development are multivalent, which represents a difficulty for the standard assays commonly used to characterize the critical quality attributes of monovalent formulations. Here, we present a multiplexed analytical tool with nucleic acid microarray technology using the VaxArray platform that measures the identity and quantity of mono- and multivalent mixtures of naked mRNA and mRNA encapsulated in lipid nanoparticle formulations in under 2 h without any additional preparation steps, such as extraction or RT-PCR. Using a quadrivalent mixture of encapsulated mRNA constructs that encode for four unique proteins in a vaccine formulation, the VaxArray mRNA assay was demonstrated to be highly specific for each mRNA with sensitivity < 1 µg/mL. The quantification of individual mRNAs within the lipid nanoparticle mixture resulted in a precision of ≤10% RSD and an accuracy of 100 ± 9%. Full article

The detection of biomarkers (such as DNA, RNA, and protein) plays a vital role in medical diagnosis. The CRISPR-based biosensors utilize the CRISPR/Cas system for biometric recognition of targets and use biosensor strategy to read out biological signals without the employment of professional operations. Consequently, the CRISPR-based biosensors demonstrate great potential for the detection of biomarkers with high sensitivity and specificity. However, the signal readout still relies on specialized detectors, limiting its application in on-site detection for medical diagnosis. In this review, we summarize the principles and advances of the CRISPR-based biosensors with a focus on medical diagnosis. Then, we review the advantages and progress of CRISPR-based naked eye biosensors, which can realize diagnosis without additional detectors for signal readout. Finally, we discuss the challenges and further prospects for the development of CRISPR-based biosensors. Full article

Coloration is a crucial trait that allows species to adapt and survive in different environments. Wild boars exhibit alternating black (dark) and yellow (light) longitudinal stripes on their back during their infancy (juvenile stripes), and as adults, they transform into uniform wild-type coat color. Aiming to record the procedure of juvenile stripes disappearing, piglets (WD) with juvenile stripes were produced by crossing a wild boar with Duroc sows, and photos of their coat color were taken from 20 d to 220 d. The pigments in the hairs from the black and yellow stripes were determined. Furthermore, the differentially expressed genes between the black and yellow stripes were investigated in 5 WD with the age of 30 d using whole-transcriptome sequencing to explore the genetic mechanism of the juvenile stripes. The juvenile stripes started to disappear at about 70 d, and stripes were not distinguished with the naked eye at about 160 d; that is, the juvenile stripe completely disappeared. A hotspot of a differentially expressing (DE) region was found on chromosome 13, containing/covering 2 of 13 DE genes and 8 of 10 DE lncRNAs in this region. A network among ZIC4, ssc-miR-532-3p, and ENSSSCG00000056225 might regulate the formation of juvenile stripes. Altogether, this study provides new insights into spatiotemporal coat color pattern. Full article

Therapeutic needs for hair loss are intended to find small interfering ribonucleic acid (siRNA) therapeutics for breakthrough. Since naked siRNA is restricted to meet a druggable target in clinic,, delivery systems are indispensable to overcome intrinsic and pathophysiological barriers, enhancing targetability and persistency to ensure safety, efficacy, and effectiveness. Diverse carriers repurposed from small molecules to siRNA can be systematically or locally employed in hair loss therapy, followed by the adoption of new compositions associated with structural and environmental modification. The siRNA delivery systems have been extensively studied via conjugation or nanoparticle formulation to improve their fate in vitro and in vivo. In this review, we introduce clinically tunable siRNA delivery systems for hair loss based on design principles, after analyzing clinical trials in hair loss and currently approved siRNA therapeutics. We further discuss a strategic research framework for optimized siRNA delivery in hair loss from the scientific perspective of clinical translation. Full article

mRNA vaccines were successfully developed and approved for emergency use to fight coronavirus disease 2019. However, the effect of DNA vaccines against SARS-CoV-2 is considerably lower than that of mRNA vaccines. A pyro-drive jet injector (PJI) efficiently delivers plasmid DNA intradermally into animal models. Here, we compared the immunogenic potential of DNA and mRNA vaccines in mice using the same platform. PJI was used to deliver naked mRNA and pDNA and their efficacy in inducing antigen expression and immune responses was assessed. Our results showed that PJI efficiently delivered mRNA into the skin, and a smaller effective dose than that of pDNA injection was required to achieve similar levels of antigen expression. The PJI-delivered CpG-free pDNA vaccine efficiently induced antigen-specific antibody production and a cell-mediated IFN-γ response compared to the mRNA vaccine, as well as the upregulation of inflammatory cytokines (IL-6, IFN-γ, and IL-1β) in the skin and lymph nodes. However, the intradermal mRNA vaccine was significantly less immunogenic than the standard intramuscular mRNA-lipid nanoparticle vaccine, despite equivalent mRNA dosages. Improvements in lipid nanoparticle and mRNA technology have revolutionized mRNA vaccines, and DNA vaccines can be similarly modified for higher clinical efficacy. Full article

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications. Full article

Therapeutic HPV vaccines that induce potent HPV-specific cellular immunity and eliminate pre-existing infections remain elusive. Among various candidates under development, those based on DNA constructs are considered promising because of their safety profile, stability, and efficacy. However, the use of electroporation (EP) as a main delivery method for such vaccines is notorious for adverse effects like pain and potentially irreversible muscle damage. Moreover, the requirement for specialized equipment adds to the complexity and cost of clinical applications. As an alternative to EP, lipid nanoparticles (LNPs) that are already commercially available for delivering mRNA and siRNA vaccines are likely to be feasible. Here, we have compared three intramuscular delivery systems in a preclinical setting. In terms of HPV-specific cellular immune responses, mice receiving therapeutic HPV DNA vaccines encapsulated with LNP demonstrated superior outcomes when compared to EP administration, while the naked plasmid vaccine showed negligible responses, as expected. In addition, SM-102 LNP M exhibited the most promising results in delivering candidate DNA vaccines. Thus, LNP proves to be a feasible delivery method in vivo, offering improved immunogenicity over traditional approaches. Full article