Molecular Therapy. Nucleic Acids最新文献

Hunner-type interstitial cystitis (HIC) is a chronic condition marked by persistent pain and inflammation. To elucidate its immunogenetic drivers, we integrated bulk RNA sequencing and single-cell RNA sequencing datasets with targeted major histocompatibility complex (MHC) sequencing. Transcriptomic analysis revealed selective expansion of B cells and epithelial cells, with strong enrichment of Epstein-Barr virus (EBV) response signatures. CellChat and NicheNet modeling uncovered bidirectional communication wherein B cells secrete IL-1β, FGF2, LIF, and TNFSF9, activating prostaglandin synthesis, matrix metalloproteinases, and stress genes in epithelial cells. In turn, epithelial BMP4, TGF-β2, and SHH modulate B cell survival. SCENIC regulatory network analysis identified IRF8 as the top B cell regulator; its regulon controls HLA-DQB1, CD40, and CIITA, linking EBV latency to heightened antigen presentation. Among differentially expressed genes, HLA-DQB1 was the most strongly induced in EBV⁺ HIC (∼1,000-fold), emerged as the most frequently mutated gene in targeted MHC sequencing, and ranked as a high-confidence IRF8 target. Notably, the evolutionarily constrained variant rs1049133 (A>G) lies within a low-entropy HLA-DQB1 domain, underscoring functional importance. Our integrated analysis supports a model where IRF8-driven, EBV-infected B cells perpetuate HIC via variant HLA-DQB1-mediated antigen presentation and epithelial cytokine loops, highlighting a tractable axis for precision therapy.

Messenger RNA (mRNA) vaccines have demonstrated significant potential in cancer immunotherapy by activating both innate and adaptive immunity. However, the detailed cellular and molecular dynamics underpinning these systemic immune responses remain incompletely understood. In this study, we characterized the systemic immune landscape following human papillomavirus (HPV)-targeted mRNA-lipid nanoparticle (LNP) vaccination using single-cell RNA sequencing (scRNA-seq) in a murine model of HPV-positive head and neck squamous cell carcinoma (HNSCC). Our study revealed a coordinated remodeling of the systemic immune landscape, involving the tumor microenvironment (TME), tumor-draining lymph nodes (TDLNs), spleen, and blood. Notably, we pioneered a distinct interferon-stimulated gene (ISG) signature across multiple lymphoid subsets in TDLNs, driven by the LNP component, which contributed to rapid, non-antigen-specific immune activation. Additionally, HPV mRNA-LNP vaccination induced an antigen-specific cycling burst of immune cells that mediated tumor control through a systemic coordination of multi-directional differentiation into anti-tumor cell compositions. These findings enhance our understanding of how mRNA-LNP vaccination orchestrates systemic anti-tumor responses and highlight the therapeutic potential of targeting ISG-expressing and cycling immune cells to improve vaccine efficacy, paving the way for future clinical applications in HPV-related cancers.

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by therapy-resistant glioma stem-like cells (GSCs) and extensive infiltration into surrounding brain tissue. MicroRNAs (miRNAs) are pleiotropic post-transcriptional regulators of oncogenic pathways, but their tumor-suppressive function is frequently lost in GBM. This study explores a multimodal therapeutic approach by restoring a combination of miRNAs to exploit their synergistic effects against GBM. Using patient-derived GBM cells cultured under stem cell-permissive conditions, we demonstrate that miRNA restoration reduces tumor growth, limits invasiveness and stemness, and enhances sensitivity to temozolomide (TMZ). In vivo studies in an orthotopic xenograft mouse model of GBM confirm the therapeutic efficacy and low toxicity of the nanoformulated miRNAs, following local injection. Multi-omics and computational analyses on different GBM subtypes reveal that these miRNAs synergistically suppress tumor-promoting extracellular matrix interactions, particularly through the collagen pathway, and downregulate genes associated with GBM progression. The genes downregulated by the miRNAs correlate with glioma grade and poor patient prognosis, further underscoring their therapeutic potential. These findings highlight the promise of combinatorial miRNA therapy as a novel strategy for GBM treatment and suggest new molecular targets for future diagnostic and therapeutic developments.

The mdx52 mouse model exhibits a common mutation profile associated with brain involvement in Duchenne muscular dystrophy (DMD), characterized by heightened anxiety, fearfulness, and impaired associative fear learning. Deletion of exon 52 disrupts the expression of two dystrophins found in the brain (Dp427 and Dp140) and is eligible for therapeutic exon-skipping strategies. We previously demonstrated that a single intracerebroventricular administration of an antisense oligonucleotide (ASO) targeting exon 51 of the Dmd gene could restore 5%-15% of Dp427 expression. This treatment reduced anxiety and unconditioned fear in mdx52 mice, improved fear conditioning acquisition, and partially improved fear memory tested 24 h later. To improve the restoration of Dp427 and induce a long-lasting therapeutic effect, we employed a vectorized approach using an adeno-associated virus (AAV)-U7 small nuclear RNA vector to deliver antisense sequences to the brains of mdx52 mice. We evaluated two AAV serotypes known for their brain transduction efficiency (AAV9 and RH10) and two delivery routes, intracisterna magna and intracerebroventricular (ICV) injections, to maximize brain targeting. Based on GFP expression data, we selected the AAV9 capsid and a bilateral ICV delivery route. Using this approach, we demonstrated that ICV administration of AAV9-U7-Ex51M induced exon 51 skipping and restored Dp427 expression in the brains of adult mdx52 mice, though with significant variability among individuals. While a few mice showed high Dp427 expression levels, the average restoration was limited to approximately 6%-12%. In conclusion, inducing exon skipping in the brains of adult mdx52 mice using the vectorized AAV9-U7 approach was less effective than synthetic ASO treatment and did not improve the emotional behavior of mdx52 mice.

Epigenetic mechanisms play a crucial role in gene expression regulation during the initiation and progression of cancer. Despite this, over 600 epigenetic regulator (ER) genes, which are responsible for the reading, writing, and erasing of histone and DNA modifications, remain insufficiently characterized in the context of human cancer. In this study, we identified 272 cancer-specific ER genes that were dysregulated in cancer, as determined using a proposed dysregulation score method, based on analysis of over 19,000 paired tumor-normal human samples. Four novel dysregulated ER genes (DEGs), uniquely identified through this method, were shown to have roles in cell proliferation and invasion in melanoma cells. We proposed that loss-of-functional mutations within epigenetic domains may influence the dysregulation of ER genes. Signature scores derived from these DEGs can serve as convenient indicators of patient prognosis in different cancer types. Our findings demonstrated that DEGs in conjunction with immune checkpoints further enhance the prediction performance of the efficiency of cancer immunotherapy compared to using immune checkpoints alone, based on independent cancer cohorts. The DEG list is a valuable resource for translational cancer research, with implications for precision oncology and the development of more effective, individualized epigenetic medicines and therapy.

Several allogeneic chimeric antigen receptor (CAR) T-cell therapies in clinical trials rely on CRISPR-Cas genome editing, but the enzyme's random repair mechanism increases the risk of undesired off-target effects, challenging safe CAR T-cell generation. To address this, we developed a novel CRISPR RNA (crRNA) targeting the T-cell receptor beta constant (TRBC) gene. Combined with AsCas12a Ultra, this crRNA edits primary human T-cells via a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, significantly lowering off-target risks. During evaluation, we sequestered a unique T-cell subset with disrupted T-cell receptor (TCR), retained CD3 expression, and no in vivo alloreactivity. Termed CD3-retained, allogeneically functioning T-cells (CRAFT-cells), these cells exhibited growth kinetics comparable to unedited T-cells. When engineered with CD19- or BAFF-R-targeted CARs, CRAFT CAR T-cells showed strong antigen-specific cytotoxicity and significant ex vivo expansion compared to conventional CD3-disrupted CAR T-cells. Moreover, CRAFT CAR T-cells effectively served as effector cells for bispecific T-cell engagers (BiTEs), enabling CD3-dependent tumor cell killing. Our CRAFT crRNA platform offers a novel strategy to generate safer allogeneic CAR T-cells. The distinct properties of CRAFT CAR T-cells, combined with BiTE therapy, represent a promising and potentially more durable approach for next-generation allogeneic CAR T-cell therapies in clinical applications.

Although Parkinson's disease (PD) is primarily idiopathic, genetic mutations-accounting for approximately 5%-15% of cases with regional variability-have prompted the development of gene expression modulators, such as oligonucleotides, to target and reduce alpha-synuclein (α-syn) accumulation. However, challenges in delivering these agents to the brain have limited their therapeutic potential. This study systematically reviews the use of exosomes as delivery systems for oligonucleotides aimed at reducing α-syn aggregation in PD. A comprehensive literature search was conducted using Scopus, Embase, OVID, and ISI Web of Science databases up to January 2022, targeting in vivo studies relevant to the subject. Of 904 initial records, five eligible studies were selected. Three utilized transgenic mouse models and two used induced models to simulate PD. All reported a reduction in α-syn aggregation in the midbrain-particularly in the substantia nigra-following treatment with exosome-delivered oligonucleotides. This reduction was associated with decreased neuronal death and improved motor function. No significant toxicity or immune response was reported. Exosome-mediated oligonucleotide delivery appears to be a promising approach to reduce α-syn aggregation, protect dopaminergic neurons, and improve motor symptoms in animal models of PD.