Guohua Yu

Introduction: CRISPR–Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches. Methods: We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules—Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope—into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days. Results: Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci. Discussion: It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

Chimeric antigen receptor–natural killer (CAR-NK) cell therapy is a promising immunotherapy for hematological malignancies. While engineered interleukin15 (IL15) variants like membrane-bound IL15 (mbIL15) and the IL15/IL15Rα heterodimer (RLI) can enhance NK cell activity, their relative efficacy and safety as armor for CAR-NK cells remain unclear. This study systematically evaluated primary human CAR-NK cells co-expressing an anti-CD19 CAR (19ζ) with soluble IL15, mbIL15, or RLI. We found that 19ζ-RLI CAR-NK cells exhibited superior IL15 secretion, proliferation, cytotoxicity, and migration in vitro, and effectively controlled tumors in vivo. However, all IL15-armored constructs, particularly 19ζ-RLI, induced lethal toxicity in mice, characterized by CAR-NK hyperproliferation and elevated systemic IL15. Transcriptomic analysis revealed that this toxicity correlated with a hyperactive molecular state driven by persistent IL15 signaling. In conclusion, this study suggests that constitutive IL15 armoring can be a potent but risky strategy for enhancing CAR-NK cells, with RLI being the most potent yet toxic exemplar of this general principle. Our findings highlight the necessity of incorporating safety-optimized strategies, such as inducible cytokine expression, into the design of cytokine-armored CAR-NK therapies for clinical translation.

Ginsenoside Rg3, a rare protopanaxadiol-type saponin enriched during the heat processing of Panax ginseng, has attracted increasing attention as a multitarget anticancer agent. This systematic review examines the anticancer potential of Rg3 through comprehensive searches of the PubMed and Web of Science databases, with a focus on peer-reviewed preclinical and clinical studies. The therapeutic efficacy of Rg3 is critically influenced by its stereochemical configuration, concentration-dependent bidirectional regulation, and pharmacokinetic constraints, including poor oral bioavailability, rapid clearance, and gut microbiota-mediated metabolism. Nanocarrier-based and targeted delivery systems have substantially improved its pharmacokinetic profile and tumor accumulation, supporting its further development for anticancer applications. Within this pharmacological context, Rg3 exhibits broad-spectrum anticancer activity across multiple solid tumors, including hepatocellular carcinoma, melanoma, lung, ovarian, breast, colon, gastric, and prostate cancers, as well as osteosarcoma, renal cancer, lung adenocarcinoma, glioblastoma, gallbladder, nasopharyngeal, cervical, and pancreatic cancers, and the hematological malignancy multiple myeloma. Mechanistically, Rg3 suppresses cancer progression through coordinated regulation of proliferation, apoptosis, autophagy, ferroptosis, angiogenesis, epithelial–mesenchymal transition, cancer stemness, immune evasion, and redox homeostasis, primarily involving the PI3K/AKT/mTOR, NF-[Formula: see text]B, MAPK, Wnt/[Formula: see text]-catenin, EGFR, and p53 pathways. These effects reflect transferable network-level mechanisms rather than tumor type-restricted actions. Moreover, Rg3 demonstrates synergistic effects with chemotherapy, radiotherapy, targeted therapy, and immunotherapy, while reversing drug resistance and attenuating treatment-related toxicity in multiple cancer models and clinical settings. Overall, this review systematically integrates current evidence on the pharmacokinetics, anticancer spectrum, molecular mechanisms, synergistic combinations, immunomodulatory effects, and clinical applications of Rg3, providing a concise framework for the rational development of Rg3-based combination strategies in precision cancer therapy.

Neuroinflammation is a significant driving force in the pathogenesis and progression of central nervous system (CNS) disorders. Polymethoxylated flavones (PMFs), the key lipid-soluble constituents in Citri Reticulatae Pericarpium (CRP), exhibit excellent blood–brain barrier permeability and anti-inflammatory properties, holding therapeutic potential for CNS disorders. However, the specific bioactive components and therapeutic effects of PMFs in treating CNS disorders are not well understood. This study employed a comprehensive sequential metabolism approach to elucidate the dynamic biotransformation of PMFs in vivo and identified seven brain-targeting components. Subsequently, network pharmacology and experimental validation were utilized to explore the potential mechanisms of PMFs. The results suggested that PMFs have potential therapeutic value for Alzheimer’s disease (AD)-like mice, with the inhibition of neuroinflammation likely being a key mechanism of their anti-AD effects. Notably, sinensetin, tangeretin, nobiletin, and 3,5,6,7,8,3′,4′-heptamethoxyflavone were identified as potent neuroinflammatory inhibitors. This research elucidated the chemical and therapeutic foundations of PMFs, indicating their potential as treatments or nutritional supplements for AD prevention and treatment. Moreover, the integrated triad approach of sequential metabolism, network pharmacology, and experimental validation may serve as a promising strategy for screening bioactive compounds in herbs or functional foods, as well as for elucidating their therapeutic mechanisms.

Cancer remains a major global health challenge, which drives the ongoing search for effective and less toxic treatment options. Due to its demonstrated anticancer properties, Artesunate (ART), a well-established antimalarial agent, has gained increasing attention as a promising candidate for oncological applications. This systematic review provides a comprehensive evaluation of ART’s therapeutic potential by examining its anticancer efficacy, underlying molecular mechanisms, synergistic capacity, and pharmacological toxicity. An extensive search of the PubMed and Web of Science databases identified relevant peer-reviewed experimental and clinical studies that investigated ART’s anticancer activity. The data were systematically extracted with an emphasis on research methodologies, treatment regimens, and mechanistic pathways. Evidence from in vitro and in vivo studies confirms ART’s broad efficacy against a range of malignancies, including hematological cancers such as lymphoma, acute myeloid leukemia, and multiple myeloma, and various solid tumors such as lung, pancreatic, colorectal, hepatocellular, breast, ovarian, bladder, gastric, cervical, glioblastoma, melanoma, retinoblastoma, and esophageal cancers. ART exerts its anticancer effects through multiple pathways, including ROS-mediated programmed cell death, ferroptosis induction, mitochondrial dysfunction, the inhibition of proliferation, and the disruption of key signaling networks such as NF-κB, STAT3, and Wnt/β-catenin cascades. Additionally, ART has been shown to enhance the efficacy of conventional chemotherapeutic agents like cisplatin and gemcitabine while also reducing associated toxicities and overcoming drug resistance. These attributes highlight ART’s considerable potential as a versatile anticancer agent that exhibits multiple — mechanisms of action and favorable compatibility with existing therapies. However, further rigorous clinical studies are essential to fully establish its therapeutic utility and facilitate its integration into modern oncology practice.

Chimeric antigen receptor T (CAR-T) cell targeting CD19 antigen has achieved exhilarative clinical efficacy in B-cell malignancies. However, challenges still remain for the currently approved anti-CD19 CAR-T therapies, including high recurrence rates, side effects and resistance. Herein, we aim to explore combinatorial therapy by use of anti-CD19 CAR-T immunotherapy and gallic acid (GA, an immunomodulatory natural product) for improving treatment efficacy. We assessed the combinatorial effect of anti-CD19 CAR-T immunotherapy with GA in cell models and a tumor-bearing mice model. Then, the underlying mechanism of GA on CAR-T cells were investigated by integrating network pharmacology, RNA-seq analysis and experimental validation. Furthermore, the potential direct targets of GA on CAR-T cells were explored by integrating molecular docking analysis with surface plasmon resonance (SPR) assay. The results showed that GA significantly enhanced the anti-tumor effects, cytokine production as well as the expansion of anti-CD19 CAR-T cells, which may be mainly through the activation of IL4/JAK3-STAT3 signaling pathway. Furthermore, GA may directly target and activate STAT3, which may, at least in part, contribute to STAT3 activation. Overall, the findings reported here suggested that the combination of anti-CD19 CAR-T immunotherapy with GA would be a promising approach to increase the anti-lymphoma efficacy.

Screening functional food ingredients (FFI) from medicinal and edible plants (MEP) has still remained a great challenge due to the complexity of MEP and its obscure function mechanisms. Herein, an integrated strategy based on sequential metabolites identification approach, network pharmacology, molecular docking, and surface plasmon resonance (SPR) analysis was proposed for quickly identifying the active constituents in MEP. First, the sequential biotransformation process of MEP, including intestinal absorption and metabolism, and hepatic metabolism, was investigated by oral gavage, and intestinal perfusion with venous sampling method. Then the blood samples were analyzed by UPLC-Q Exactive Orbitrap HRMS. Second, the network pharmacology approach was used to explore the potential targets and possible mechanisms of the in vivo metabolites of MEP. Third, molecular docking and SPR approaches were used to verify the specific interactions between protein targets and representative ingredients. The proposed integrated strategy was successfully used to explore the heptoprotective components and the underlying molecular mechanism of Paeoniae Radix Alba (PRA). A total of 44 compounds were identified in blood samples, including 17 porotypes and 27 metabolites. The associated metabolic pathways were oxidation, methylation, sulfation, and glucuronidation. After further screening, 31 bioactive candidates and 377 related targets were obtained. In addition, the bioactive components contained in PRA may have therapeutic potentials for non-alcoholic fatty liver disease (NAFLD). The above results demonstrated the proposed strategy may provide a feasible tool for screening FFI and elaborating the complex function mechanisms of MEP.