Current Gene Therapy - Volume 25, Issue 5, 2025

Significant variations in immune profiles across different age groups manifest distinct clinical symptoms and prognoses in Coronavirus Disease 2019 (COVID-19) patients. Predominantly, severe COVID-19 cases that require hospitalization occur in the elderly, with the risk of severe illness escalating with age among young adults, children, and adolescents.

This study aimed to delineate the unique immune characteristics of COVID-19 across various age groups and evaluate the feasibility of detecting COVID-19-induced immune alterations through peripheral blood analysis.

By employing a machine learning approach, we analyzed gene expression data from nasopharyngeal and peripheral blood samples of COVID-19 patients across different age brackets. Nasopharyngeal data reflected the immune response to COVID-19 in the upper respiratory tract, while peripheral blood samples provided insights into the overall immune system status. Both datasets encompassed COVID-19 patients and healthy controls, with patients divided into children, adolescents, and adult age groups. The analysis included the expression levels of 62,703 genes per patient. Then, 9 feature-sequencing methods (least absolute shrinkage and selection operator, light gradient boosting machine, Monte Carlo feature selection, random forest, ridge regression, adaptive boosting, categorical boosting, extremely randomized trees, and extreme gradient boosting) were employed to evaluate the association of the genes with COVID-19. Key genes were then utilized to develop efficient classification models.

The findings identified specific markers: insulin-like growth factor binding protein 3 (downregulated in the peripheral blood of COVID-19 patients), interferon alpha-inducible protein 27 (upregulated), and SERPING1 (upregulated in nasopharyngeal tissues). In addition, fibulin-2 was downregulated in adolescent patients, but upregulated in the other groups, while epoxide hydrolase 3 was upregulated in healthy controls, but downregulated in children and adolescents.

This study offers valuable insights into the local and systemic immune responses of COVID-19 patients across age groups, aiding in identifying potential therapeutic targets and formulating personalized treatment strategies.

Plasmalogens, the primary phospholipids in the brain, possess intrinsic antioxidant properties and are crucial components of the myelin sheath surrounding neuronal axons. While their neuroprotective effects have been demonstrated in Alzheimer's disease, their potential benefits in spinal cord injury remain unexplored. This study investigates the reparative effects of plasmalogens on spinal cord injury and the underlying mechanisms.

In vitro, we developed dorsal root ganglion (DRG) and RAW 264.7 cell models under high-reactive oxygen species (ROS) conditions to assess ROS levels, neuronal damage, and inflammatory microenvironment changes before and after plasmalogen application. In vivo, we used a complete mouse spinal cord transection model to evaluate changes in ROS levels, neuronal demyelination, and apoptosis following plasmalogen treatment. Additionally, we assessed sensory and motor function recovery and investigated the regulatory effects of plasmalogens on the AKT/mTOR signaling pathway.

In high-ROS cell models, plasmalogens protected DRG neurons (TUJ-1) from axonal damage and modulated the proinflammatory/anti-inflammatory balance in RAW 264.7 cells. In vivo, plasmalogens significantly reduced ROS levels, improved the immune microenvironment, decreased the proinflammatory (iNOS)/anti-inflammatory (ARG-1) ratio, lowered neuronal (TUJ-1) apoptosis (Caspase-3, BAX), and reduced axonal degeneration while promoting myelin (MBP) regeneration, indicating a neuroprotective effect. These findings are linked to the activation of the AKT/mTOR signaling pathway.

Plasmalogens reduce ROS levels and regulate inflammation-induced damage, contributing to neuroprotection. This study reveals that plasmalogens promote remyelination, reduce axonal degeneration and neuronal apoptosis, and—used here for the first time in spinal cord injury repair—may protect neurons by reducing ROS levels and activating the AKT/mTOR signaling pathway.

The clinical translation of chitosan-based formulations for siRNA delivery has been partially limited by their poor stability/solubility at physiological conditions, although they have good biocompatibility and cost-effectiveness.

In this study, the chitosan was O-substituted with diisopropylethylamine (DIPEA) groups, which improved its solubility at pH 7.4 by increasing the degree of ionization and enhanced the ability of chitosan to load siRNA at very low amine/phosphate (N/P) ratios. The O-DIPEAchitosan/siRNA nanopolyplexes were self-assembled by complexation and presented positive Zeta potentials (ζ = +8 to +10 mV), spherical-like morphology, 200-300 nm size, low polydispersity index (PDI < 0.2), and were able to protect the siRNA from degradation by RNAse. Also, the resistance to albumin-induced disassembly and aggregation revealed both good structural and colloidal stabilities of the siRNA nanopolyplexes.

The nanopolyplexes displayed low cytotoxicities in RAW 264.7 macrophages and were successfully uptaken by both macrophages and HeLa cells achieving internalization efficiency similar to Lipofectamine. A positive correlation was observed between the physicochemical properties of the siRNA nanocarrier and its transfection efficiency.

A knockdown of about 60-70% of tumor necrosis factor alpha (TNFα) was reached in lipopolysaccharide-stimulated macrophages treated with O-DIPEA-chitosan/siTNFα nanopolyplexes. Overall, the results confirmed that O-DIPEA chitosans are promising carriers for siRNA delivery.

Liposomes are versatile delivery systems for encapsulating small interfering RNAs (siRNAs) because they enhance cellular uptake and gene silencing. This study compares the new liposome formula to commercial lipofectamine in delivering siRNAs targeting hepatic carcinoma genes, focusing on HNF4-α and PFKFB4.

Flow cytometry and confocal microscopy revealed efficient internalization of PE-Rhod-B labeled lipoplexes in HepG2 cells, while cytotoxicity assays demonstrated significant reductions in cell viability, particularly with siHNF4-α and siPFKFB4.

The newly formulated liposomes showed superior efficacy, achieving nearly 93% cytotoxicity at 100 nM, compared to just 50% with lipofectamine at the same concentration. Furthermore, real-time PCR confirmed that the liposome-encapsulated siHNF4-α reduced HNF4-α mRNA expression by tenfold at 100 nM, compared to a twofold reduction with lipofectamine at 200 nM. Similarly, siPFKFB4 delivered via liposomes showed a dose-dependent 35-fold reduction in PFKFB4 mRNA expression at 100 nM, outperforming the maximum reduction achieved by lipofectamine. The IC50 values for all siRNA treatment groups were significantly lower when using the liposome formula, reflecting improved delivery efficiency.

These results demonstrate the potential of liposome formulations for therapeutic siRNA delivery. The encapsulation enhances cellular uptake and gene silencing efficiency, making the liposome formula a promising candidate for targeted gene therapy in hepatic carcinoma. Further research should explore it’s in vivo biodistribution and potential combination therapies.

Fibrosis refers to abnormal deposition of extracellular matrix, which leads to organ dysfunction. Metabolic alterations, especially enhanced glycolysis and suppressed fatty acid oxidation, are recognized as an essential pathogenic process of fibrosis. Recently, several reports indicate that the changes in microbiota composition are associated with metabolic disorders, suggesting microbes may contribute to organ fibrosis by regulating metabolic processes.

In this study, microbial reannotation was carried out on the RNA-seq data of fibrotic organs. Then, the microbial composition differences among healthy and fibrotic organ samples were determined by alpha and beta diversity analysis. Common and specific microbial markers of fibrosis were also identified by LEfSe. After that, the correlation analysis of the characteristic microbe- gene-functional pathway was conducted to confirm the effects of microbes on host metabolism.

The results showed that the microbial composition significantly differed between healthy and diseased organs. Besides, the common characteristic microbes interacted closely with each other and contributed to fibrosis through symbiosis or inhibition. The largest proportion in fibrosis organs was Proteobacteria, which was the main source of pathogenic microbes.

Further study found that the metabolic alteration driven by common and special characteristic microbes in fibrotic organs focused on the processes related to glycolysis and fatty acid metabolism.

The global prevalence of rheumatoid arthritis (RA) is on the rise. Numerous studies have demonstrated the potential of stem cell-based therapies in RA treatment. Experimental evidence suggests that preconditioning enhances the regenerative capabilities of stem cells compared to their unconditioned counterparts.

This study aimed to evaluate whether adipose-derived stem cells (ADSCs) preconditioned with green tea epigallocatechin gallate (EGCG) and miR-92a exhibit superior therapeutic effects in RA compared to unconditioned ADSCs.

Both in vitro and in vivo models were employed. In the cellular model, ADSCs were preconditioned with EGCG and miR-92a. In the animal model, male Wistar rats were used, and RA was induced using the collagen-induced arthritis (CIA) model. Following RA induction, the animals were divided into six groups: Sham (healthy rats), RA (RA-induced rats), RA+ADSC (RA-induced rats receiving unconditioned ADSCs), RA+E-ADSC (RA-induced rats receiving EGCG-preconditioned ADSCs), RA+mic-ADSC (RA-induced rats receiving miR-92a mimic-preconditioned ADSCs), and RA+inh-ADSC (RA-induced rats receiving miR-92a inhibitor-preconditioned ADSCs).

In the cellular model, preconditioning with EGCG and miR-92a activated the CXCR4/p-Akt signaling pathway, thereby enhancing ADSC viability. In the animal model, RA induction caused several joint pathologies, including hind paw swelling, disrupted bone metabolism, immune cell infiltration, increased expression of IL-17, KLF4, and IL-6, as well as cartilage degradation. While transplantation of unconditioned ADSCs modestly improved these pathological features, the administration of E-ADSCs and mic-ADSCs significantly ameliorated these conditions in RA rats. Conversely, the therapeutic effects of E-ADSCs and mic-ADSCs were attenuated by the transplantation of inh-ADSCs.

The therapeutic effects of E-ADSCs and mic-ADSCs in RA were strongly associated with the modulation of the KLF4/IL-17/MMP-2 axis. These findings suggest that ADSCs preconditioned with EGCG and miR-92a hold considerable clinical promise for the treatment of RA.

Antimicrobial peptides (AMPs), unlike antibiotics, are encoded in genomes. AMPs are exported from the cell after expression and translation. In the case of bacteria, the exported peptides target other microbes to give the producing bacterium a competitive edge. While AMPs are sought after for their similar antimicrobial activity to traditional antibiotics, it is difficult to predict which combinations of amino acids will confer antimicrobial activity. Many computer algorithms have been designed to predict whether a sequence of amino acids will exhibit antimicrobial activity, but the vast majority of validated AMPs in databases are still of eukaryotic origin. This defies common sense since the vast majority of life on Earth is prokaryotic.

The antimicrobial peptide pipeline, presented here, is a bacteria-centric AMP predictor that predicts AMPs by taking design inspiration from the sequence properties of bacterial genomes with the intention to improve the detection of naturally occurring bacterial AMPs. The pipeline integrates multiple concepts of comparative biology to search for candidate AMPs at the primary, secondary, and tertiary peptide structure levels.

Results showed that the antimicrobial peptide pipeline identifies known AMPs that are missed by state-of-the-art AMP predictors and that the pipeline yields more AMP candidates from real bacterial genomes than from fake genomes, with the rate of AMP detection being significantly higher in the genomes of six nosocomial pathogens than in the fake genomes.

This bacteria-centric AMP pipeline enhances the detection of bacterial AMPs by incorporating sequence properties unique to bacterial genomes. It complements existing tools, addressing gaps in AMP detection and providing a promising avenue for discovering novel antimicrobial peptides.

Cancer-Associated Fibroblasts (CAFs) constitute a heterogeneous group of cells critical for the remodeling of the tumor microenvironment (TME). Given their significant impact on tumor progression, particularly in skin cancers, a deeper understanding of their characteristics and functions is essential.

This study employed a single-cell transcriptomic analysis to explore the diversity of CAFs within three major types of skin cancer: basal cell carcinoma, melanoma, and head and neck squamous cell carcinoma. We applied analytical techniques, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), pseudotime tracking, metabolic profiling, and stemness assessment to delineate and define the functional attributes of identified CAF subgroups.

Our analysis successfully delineated nine distinct CAF subgroups across the studied tumor types. Of particular interest, we identified a novel CAF subtype, designated as C0, exclusive to basal cell carcinoma. This subtype exhibits phenotypic traits associated with invasive and destructive capabilities, significantly correlating with the progression of basal cell carcinoma. The identification of this subgroup provides new insights into the role of CAFs in cancer biology and opens avenues for targeted therapeutic strategies.

A pan-cancer analysis was performed on three cancers, BCC, MA, and HNSCC, focusing on tumor fibroblasts in TME. Unsupervised clustering categorized CAF into nine subpopulations, among which the C0 subpopulation had a strong correspondence with BCC-CAF and an invasive-destructive-related phenotype.