Rationale: Circular RNA (circRNA) has gained attention as a promising platform for mRNA vaccines due to its stability, sustained protein expression, and intrinsic immunostimulatory properties. This study aimed to design and optimize a circRNA cancer vaccine platform by screening for efficient internal ribosome entry sites (IRES) and enhancing circRNA translation efficiency for improved cancer immunotherapy.

Circular RNAs (circRNAs) have emerged as a prominent class of covalently closed single-stranded RNA molecules that exhibit tissue-specific expression and potential as biomarkers in extracellular vesicles (EVs) derived from liquid biopsies. Still, their characteristics and applications in EVs remain to be unveiled. We performed a comprehensive analysis of EV-derived circRNAs (EV-circRNAs) using transcriptomics data obtained from 1082 human body fluids, including plasma, urine, cerebrospinal fluid (CSF), and bile. Our validation strategy utilized RT-qPCR and RNA immunoprecipitation assays, complemented by computational techniques for analyzing EV-circRNA features and RNA-binding protein interactions. We identified 136,327 EV-circRNAs from various human body fluids. Significantly, a considerable amount of circRNAs with a high back-splicing ratio are highly enriched in EVs compared to linear RNAs. Additionally, we discovered brain-specific circRNAs enriched in plasma EVs and cancer-associated EV-circRNAs linked to clinical outcomes. Moreover, we demonstrated that EV-circRNAs have the potential to serve as biomarkers for evaluating immunotherapy efficacy in non-small cell lung cancer (NSCLC). Importantly, we identified the involvement of RBPs, particularly YBX1, in the sorting mechanism of circRNAs into EVs. This study unveils the extensive repertoire of EV-circRNAs across human biofluids, offering insights into their potential as disease biomarkers and their mechanistic roles within EVs. The identification of specific circRNAs and the elucidation of RBP-mediated sorting mechanisms open new avenues for the clinical application of EV-circRNAs in disease diagnostics and therapeutics.

Purpose: In China, many patients with hepatocellular carcinoma (HCC) are diagnosed at an advanced stage. Several studies have shown that triple therapy [transarterial chemoembolization (TACE) combined with tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs)] is beneficial for patient survival. In this study, we aimed to evaluate the efficacy of triple therapy (TACE + TKIs + ICIs) for unresectable HCC (uHCC) and the conversion rate of surgical resection (SR). The primary endpoints were objective response rate (ORR) and disease control rate (DCR) based on the modified Response Evaluation Criteria in Solid Tumors (mRECIST) and RECIST v1.1 and adverse events (AEs), while the secondary endpoint was the conversion rate of patients with uHCC treated with triple therapy followed by SR. Patients and Methods: Forty-nine patients with uHCC who received triple therapy at Fujian Provincial Hospital between January 2020 and June 2022 were retrospectively included. The treatment efficacy, SR conversion rate, and associated AEs were recorded. Results: Among the 49 patients enrolled, the ORRs assessed by mRECIST and RECIST v1.1 were 57.1% (24/42) and 14.3% (6/42), respectively, and the DCRs were 92.9% (39/42) and 88.1% (37/42), respectively. Seventeen (34.7%) patients met the criteria for resectable HCC and underwent resection. The median interval between the start of triple therapy and resection was 113.5 days (range 94.75 to 182 d), and the median number of TACE was 2 (range 1 to 2.5). The patients did not achieve median overall survival or median progression-free survival. Treatment-related AEs occurred in 48 (98%) patients, and 18 (36.7%) patients had grade ≥ 3 AEs. Conclusion: Triple combination therapy resulted in a relatively high ORR and conversion resection rate following uHCC treatment.

Background: Drug-eluting bead transarterial chemoembolization (DEB-TACE) has good efficacy in the treatment of unresectable hepatocellular carcinoma (uHCC), with a relatively high objective response rate (ORR) compared to conventional transarterial chemoembolization (cTACE). This study aimed to evaluate the safety and medium-term clinical efficacy of DEB-TACE combined with lenvatinib (LEN) plus PD-1 inhibitors as a triple therapy for the treatment of uHCC. Methods: Data of patients with uHCC who received triple therapy of DEB-TACE combined with LEN plus PD-1 inhibitors from January 2019 to June 2021 were analyzed retrospectively. The study endpoints were ORR, progression-free survival (PFS), and treatment-related adverse events based on the modified Response Evaluation Criteria in Solid Tumors (mRECIST). Results: Thirty-five patients were included in this study, with a median follow-up period of 15 months. The median cycle of DEB-TACE was 1, while that of all forms of TACE procedures per patient was 2. The median administration time of LEN was 7 months, and the median number of PD-1 inhibitor treatment was 4 cycles. The ORR based on mRECIST was 82.9%, disease control rate was 91.4%, and the median time to response was 7 weeks. Among these, the ORR of Barcelona Clinic Liver Cancer (BCLC) stage A reached 100%, while that of BCLC stages B and C reached 84.6% and 78.9%, respectively. The median PFS was 9 months; the mOS was not reached. Fourteen patients (40%) successfully underwent downstaging conversion and surgical resection, 32 patients (91.4%) experienced treatment-related adverse events, and no grade 5-related adverse reactions occurred. Conclusion: DEB-TACE combined with LEN and PD-1 inhibitors has a high ORR and surgical conversion rate in the treatment of uHCC tumors, and the toxicity and side effects were tolerable.

Antithrombin (AT) is the main physiological anticoagulant involved in hemostasis. Hereditary AT deficiency is a rare autosomal dominant thrombotic disease mainly caused by mutations in SERPINC1, which was usually manifested as venous thrombosis and pulmonary embolism. In this study, we analyzed the clinical characteristics and screened for mutant genes in two pedigrees with hereditary AT deficiency, and the functional effects of the pathogenic mutations were evaluated. Candidate gene variants were analyzed by next-generation sequencing to screen pathogenic mutations in probands, followed by segregation analysis in families by Sanger sequencing. Mutant and wild-type plasmids were constructed and transfected into HEK293T cells to observe protein expression and cellular localization of SERPINC1. The structure and function of the mutations were analyzed by bioinformatic analyses. The proband of pedigree A with AT deficiency carried a heterozygous frameshift mutation c.1377delC (p.Asn460Thrfs*20) in SERPINC1 (NM000488.3), a 1377C base deletion in exon 7 resulting in a backward shift of the open reading frame, with termination after translation of 20 residues, and a different residue sequence translated after the frameshift. Bioinformatics analysis suggests that the missing amino acid sequence caused by the frameshift mutation might disrupt the disulfide bond between Cys279 and Cys462 and affect the structural function of the protein. This newly discovered variant is not currently included in the ClinVar and HGMD databases. p.Arg229* resulted in a premature stop codon in exon 4, and bioinformatics analysis suggests that the truncated protein structure lost its domain of interaction with factor IX (Ala414 site) after the deletion of nonsense mutations. However, considering the AT truncation protein resulting from the p.Arg229* variant loss a great proportion of the molecule, we speculate the variant may affect two functional domains HBS and RCL and lack of the corresponding function. The thrombophilia and decreased-AT-activity phenotypes of the two pedigrees were separated from their genetic variants. After lentiviral plasmid transfection into HEK293T cells, the expression level of AT protein decreased in the constructed c.1377delC mutant cells compared to that in the wild-type, which was not only reduced in c.685C > T mutant cells but also showed a significant band at 35 kDa, suggesting a truncated protein. Immunofluorescence localization showed no significant differences in protein localization before and after the mutation. The p.Asn460Thrfs*20 and p.Arg229* variants of SERPINC1 were responsible for the two hereditary AT deficiency pedigrees, which led to AT deficiency by different mechanisms. The p.Asn460Thrfs*20 variant is reported for the first time.

The integration of photoacoustic imaging (PAI) and photothermal therapy (PTT) within the second near-infrared (NIR-II) window, offering a combination of high-resolution imaging and precise non-invasive thermal ablation, presents an attractive opportunity for cancer treatment. Despite the significant promise, the development of this noninvasive phototheranostic nanomedicines encounters challenges that stem from tumor thermotolerance and limited therapeutic efficacy. In this contribution, we designed an amphiphilic semiconducting polymer brush (SPB) featuring a thermosensitive carbon monoxide (CO) donor (TDF-CO) for NIR-II PAI-assisted gas-augmented deep-tissue tumor PTT. TDF-CO nanoparticles (NPs) exhibited a powerful photothermal conversion efficiency (43.1%) and the capacity to trigger CO release after NIR-II photoirradiation. Notably, the liberated CO not only acts on mitochondria, leading to mitochondrial dysfunction and promoting cellular apoptosis but also hinders the overexpression of heat shock proteins (HSPs), enhancing the tumor’s thermosensitivity to PTT. This dual action accelerates cellular thermal ablation, achieving a gas-augmented synergistic therapeutic effect in cancer treatment. Intravenous administration of TDF-CO NPs in 4T1 tumor-bearing mice demonstrated bright PAI signals and remarkable tumor ablation under 1064 nm laser irradiation, underscoring the potential of CO-mediated photothermal/gas synergistic therapy. We envision this tailor-made multifunctional NIR-II light-triggered SPB provides a feasible approach to amplify the performance of PTT for advancing future cancer phototheranostics.