Inflammation and fibrosis in human liver are often precursors to hepatocellular carcinoma (HCC), yet none of them is easily modeled in animals. We previously generated transgenic mice with hepatocyte-specific expressed herpes simplex virus thymidine kinase (HSV-tk). These mice would develop hepatitis with the administration of ganciclovir (GCV). However, our HSV-tk transgenic mice developed hepatitis and HCC tumor as early as six months of age even without GCV administration. We analyzed the transcriptome of the HSV-tk HCC tumor and hepatitis tissue using microarray analysis to investigate the possible causes of HCC. Gene Ontology (GO) enrichment analysis showed that the up-regulated genes in the HCC tissue mainly include the immune-inflammatory and cell cycle genes. The down-regulated genes in HCC tumors are mainly concentrated in the regions related to lipid metabolism. Gene set enrichment analysis (GSEA) showed that immune-inflammatory-related signals in the HSV-tk mice are up-regulated compared to those in Notch mice. Our study suggests that the immune system and inflammation play an important role in HCC development in HSV-tk mice. Specifically, increased expression of immune-inflammatory-related genes is characteristic of HSV-tk mice and that inflammation-induced cell cycle activation maybe a precursory step to cancer. The HSV-tk mouse provides a suitable model for the study of the relationship between immune-inflammation and HCC, and their underlying mechanism for the development of therapeutic application in the future.

β ⁶⁵⁴ -thalassemia is caused by a point mutation in the second intron (IVS-II) of the β-globin gene that activates a cryptic 3′ splice site, leading to incorrect RNA splicing. Our previous study demonstrated that when direct deletion of the β ⁶⁵⁴ mutation sequence or the cryptic 3′ splice site in the IVS-II occurs, correct splicing of β-globin mRNA can be restored. Herein, we conducted an in-depth analysis to explore a more precise gene-editing method for treating β ⁶⁵⁴ -thalassemia. A single-base substitution of the cryptic 3′ acceptor splice site was introduced in the genome of a β ⁶⁵⁴ -thalassemia mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9(Cas9)-mediated homology-directed repair (HDR). All of the HDR-edited mice allow the detection of correctly spliced β-globin mRNA. Pathological changes were improved compared with the nonedited β ⁶⁵⁴ mice. This resulted in a more than twofold increase in the survival rate beyond the weaning age of the mice carrying the β ⁶⁵⁴ allele. The therapeutic effects of this gene-editing strategy showed that the typical β-thalassemia phenotype can be improved in a dose-dependent manner when the frequency of HDR is over 20%. Our research provides a unique and effective method for correcting the splicing defect by gene editing the reactive splicing acceptor site in a β ⁶⁵⁴ mouse model.

Background/Objectives: X-linked Alport syndrome (XLAS) arises from pathogenic variants in COL4A5. Truncating variants are generally classified as severe, but whether clinically meaningful heterogeneity exists within this group remains unclear. This study aimed to establish a novel Col4a5 knock-in mouse model based on a clinical variant and to determine whether truncating mutation position influences disease severity. Methods: A de novo COL4A5 frameshift variant, c.2440delG, was identified in a patient with severe early-onset XLAS. A Col4a5-G814fs knock-in mouse was generated by CRISPR/Cas9 on the C57BL/6J inbred mouse strain background and compared with the established Col4a5-G5X nonsense model using survival analysis, serial functional measurements, kidney histopathology, transmission electron microscopy, and RNA sequencing. Results: The Col4a5-G814fs knock-in mouse was successfully generated and showed loss of glomerular α5(IV) collagen chain expression. Compared with G5X mice, G814fs mice exhibited shorter survival (median 141 vs. 161.5 days, p = 0.0004), earlier onset of proteinuria, and more severe kidney functional decline. By 16 weeks, G814fs mice also showed more severe glomerular basement membrane abnormalities and more extensive glomerulosclerosis. RNA sequencing revealed a shared inflammatory gene signature in both models, together with selective upregulation of genes related to the PPAR signaling pathway and fatty acid metabolism in G814fs kidneys. Conclusions: This study reports a novel de novo COL4A5 frameshift variant and establishes the first Col4a5-G814fs knock-in mouse model. Direct comparison with the G5X model shows that distinct truncating COL4A5 mutations can be associated with substantially different disease severity, providing a useful platform for future mechanistic and therapeutic studies in XLAS.

Background: The “hygiene hypothesis” and “farm effect” suggest that early-life microbial exposure protects against allergic diseases. Maternal low-dose lipopolysaccharide (LPS) exposure during pregnancy attenuates allergic airway inflammation in offspring, yet the underlying mechanisms remain incompletely understood. Immunoglobulin G (IgG) N-glycosylation critically regulates antibody effector functions and maternal–fetal immune transfer, but its role in transgenerational allergy protection is unknown. Objective: To investigate whether maternal low-dose LPS exposure modulates serum IgG N-glycan profiles in offspring and whether these glycomic changes are associated with protection against house dust mite (HDM)-induced allergic airway inflammation. Methods: Pregnant C57BL/6J mice received intraperitoneal LPS (7 µg/kg) or phosphate-buffered saline (PBS) on gestational day 15. Offspring were sensitized with HDM extract or PBS at 6 weeks of age. Serum IgG was purified using Protein G affinity chromatography, and N-glycans were released with PNGase F, labeled with 2-aminobenzamide, and analyzed by ultra-performance liquid chromatography (UPLC) with fluorescence detection. Twelve glycan peaks (GP1-GP12) and five derived traits (galactosylation, sialylation, monogalactosylation, digalactosylation, and bisecting glycans) were quantified. Results: HDM challenge significantly altered offspring IgG glycosylation, characterized by decreased digalactosylation, total galactosylation, and sialylation, consistent with inflammatory glycan signatures reported in other inflammatory diseases. Maternal LPS exposure alone induced similar glycomic shifts in offspring (decreased digalactosylation, total galactosylation, and sialylation). In HDM-challenged offspring, maternal LPS pretreatment produced a trend toward reduced lung inflammation without statistical significance; however, IgG glycan differences between LPS-HDM and PBS-HDM groups were non-significant. Conclusions: Maternal low-dose LPS exposure reduced offspring IgG galactosylation and sialylation. HDM challenge produced similar glycomic changes. Despite a trend toward attenuation of HDM-induced lung inflammation, maternal LPS did not restore IgG glycosylation. These findings provide first evidence linking prenatal immune stimulation to transgenerational IgG glycomic remodeling.