The effectiveness of adeno-associated virus (AAV)-based gene therapy is frequently constrained by the presence of AAV-neutralizing antibodies (NAbs). Existing detection techniques have shown inconsistencies across laboratories and cellular dependencies, challenging their universal applicability. Here, we redefine the NAb titer concept to represent the capability to neutralize a specific number of AAV virions per milliliter of serum. We present the AAV-homology-directed repair (HDR) assay, which harnesses the CRISPR-Cas9 system, offering a precise and sensitive means of detecting AAV NAbs. This assay employs a promoterless AAV HDR vector for integration into electroporated cells, facilitating the stable expression of a quantifiable fluorescent reporter and subsequent NAb titer assessment. Comparative evaluations indicated that the AAV-HDR method outperforms the traditional AAV overexpression (AAV-OE) assay regarding sensitivity and consistency. Crucially, it produced consistent outcomes across various cell lines, suggesting its potential as a universal standard for NAb titer measurement. We further confirmed the validity of the AAV-HDR titration approach by juxtaposing it with the established NT50 assay. Notably, the AAV-HDR method correlated robustly with both the AAV-OE assay and NT50 NAb titer values, and it exhibited heightened efficacy in identifying low-titer antibodies compared with the NT50 method. Given its ability to address AAV NAb detection challenges, the AAV-HDR assay holds promise for refining therapeutic strategies in gene therapy, particularly in tailoring AAV doses to neutralize preexisting NAbs.

Background CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. Results We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. Conclusions Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.

Long non-coding RNAs are thought to play a vital role in a variety of human malignancies. Studies have shown that MIR155 host gene (MIR155HG) acts as an oncogene in several cancers, but the function and its mechanism of MIR155HG in gastric cancer (GC) is still poorly understood. In this study, we determined the biological functions and underlying mechanisms of MIR155HG in GC cells. We found that expression levels of MIR155HG was increased markedly in GC patients' serum. In vitro and in vivo studies demonstrated that MIR155HG modulated the malignant phenotype of GC cells, such as cell proliferation, colony forming ability, cell migration ability, and tumor growth in nude mice. Next, our results revealed that NF-κB and STAT3 signaling pathways could be involved in regulating the malignant behavior of GC cells. Our rescue experiments showed that inhibiting NF-κB and STAT3 signaling pathways attenuated the phenotypes caused by MIR155HG overexpression. Moreover, cytotoxicity and apoptosis assays revealed overexpression of MIR155HG reduced the apotosis of GC cells induced by cisplatin and 5-FU. Together, our studies suggested that MIR155HG overexpression promoted proliferation, migration, and chemoresistance of GC cells. These results might provide a lncRNA-based target for GC treatment in future.

Purpose: Colorectal cancer (CRC) is characterized by the absence of obvious symptoms in the early stage. Due to the high rate of late diagnosis of CRC patients, the mortality rate of CRC is higher than that of other malignant tumors. Accumulating evidence has demonstrated that UBQLN1 plays an important role in many biological processes. However, the role of UBQLN1 in CRC progression is still elusive. Methods and results: we found that UBQLN1 was significantly highly expressed in CRC tissues compared with normal tissues. Enhanced/reduced UBQLN1 promoted/inhibited CRC cell proliferation, colony formation, epithelial–mesenchymal transition (EMT) in vitro, and knockdown of UBQLN1 inhibited CRC cells’ tumorigenesis and metastasis in nude mice in vivo. Moreover, the knockdown of UBQLN1 reduced the expression of c-Myc by downregulating the ERK-MAPK pathway. Furthermore, the elevation of c-Myc in UBQLN1-deficient cells rescued proliferation caused by UBQLN1 silencing. Conclusions: Knockdown of UBQLN1 inhibits the progression of CRC through the ERK-c-Myc pathway, which provides new insights into the mechanism of CRC progression. UBQLN1 may be a potential prognostic biomarker and therapeutic target of CRC.

A series of clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated protein 9(Cas9)systems have been engineered for genome editing.The most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus.However,a comparison of their detailed gene editing outcomes is still lacking.By characterizing the editing out-comes of 11 sites in human induced pluripotent stem cells(iPSCs)and K562 cells,we found that SaCas9 could edit the genome with greater efficiencies than SpCas9.We also compared the effects of spacer lengths of single-guide RNAs(sgRNAs;18-21 nt for SpCas9 and 19-23 nt for SaCas9)and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9,respectively.However,the optimal spacer length for a particular sgRNA was 18-21 nt for SpCas9 and 21-22 nt for SaCas9.Furthermore,SpCas9 exhibited a more substantial bias than SaCas9 for nonhomolo-gous end-joining(NHEJ)+1 insertion at the fourth nucleotide upstream of the protospacer adja-cent motif(PAM),indicating a characteristic of a staggered cut.Accordingly,editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide(dsODN)inser-tion or homology-directed repair(HDR)-mediated adeno-associated virus serotype 6(AAV6)donor knock-in.Finally,GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9.Our work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

Objective To investigate the effect of NAIF1 in gastric cancer cell lines MKN45. Methods We constructed pLVX-Tight-Flag-NAIF1-puro plasmid with Tet-on system. DOX was added to induce NAIF1 expression in MKN45 cells. The cells were collected at 0, 6, 12 and 24 hours after DOX addition for gene expression microarray detection and biological analysis of differentially expressed genes. qRT-PCR and Western blot were used to verify the changes in mRNA and protein levels of the selected target differential genes. Results The biological analysis of gene microarray hybridization results showed that IFIT1, IFIT2 and IFIT3 expression significantly increased at 24h, qRT-PCR also showed this change, and Western blot further verified the change in protein level. However, IFIT5 showed no significant change in mRNA and gene expression. Conclusion Over-expression of NAIF1 in gastric cancer cells can promote the expression of some immune system-related IFIT family proteins.

To investigate the expression and mechanism of LSC27A6 in papillary thyroid cancer (PTC). We analyzed the differential expression of SLC27A6 in PTC tissues and normal tissues based on the TCGA database and validated it using immunohistochemistry. Wilcoxon rank sum, chi-square test, or Fisher exact exam were used to analyze the relationship between the expression of SLC27A6 and clinicopathological information. Samples were divided into two groups according to whether BRAF was mutated or not, and Wilcoxon rank sum was used to determine whether the expression of SLC27A6 was related to BRAF mutation. The effects of SLC27A6 on the proliferation, migration, and apoptosis of PTC cells were detected by cell counting kit-8 (CCK8), colony formation assay, transwell assay, and flow cytometry. Spearman correlation analysis was used to evaluate the relationship between SLC27A6 and c-MYC. Protein expression was detected by Western blot. The expression of SLC27A6 was higher in PTC and positively correlated with N stage. SLC27A6 expression was higher in samples with BRAF mutations. Down-regulation of SLC27A6 inhibited cell proliferation, migration, and invasion and induced apoptosis. Spearman correlation analysis showed that SLC27A6 was positively correlated with c-MYC. Knockdown of SLC27A6 inhibited c-MYC expression. Our results suggest that SLC27A6 is overexpressed in PTC tissues and affects the progression of PTC by regulating c-MYC.

BACKGROUND:After repairing double-strand breaks (DSBs) caused by CRISPR-Cas9 cleavage, genomic damage, such as large deletions, may have pathogenic consequences.RESULTS:We show that large deletions are ubiquitous but are dependent on editing sites and cell types. Human primary T cells display more significant deletions than hematopoietic stem and progenitor cells (HSPCs), whereas we observe low levels in induced pluripotent stem cells (iPSCs). We find that the homology-directed repair (HDR) with single-stranded oligodeoxynucleotides (ssODNs) carrying short homology reduces the deletion damage by almost half, while adeno-associated virus (AAV) donors with long homology reduce large deletions by approximately 80%. In the absence of HDR, the insertion of a short double-stranded ODN by NHEJ reduces deletion indexes by about 60%.CONCLUSIONS:Timely bridging of broken ends by HDR and NHEJ vastly decreases the unintended consequences of dsDNA cleavage. These strategies can be harnessed in gene editing applications to attenuate unintended outcomes.

OBJECTIVE:To study the expression of LINC00673 in cervical cancer and cervical intraepithelial neoplasia (CIN) and to explore the role of LINC00673 in the development of cervical cancer.METHODS:The expression of LINC00673 in serum from cervical cancer patients, CIN patients, and healthy participants was detected by RT-qPCR. The function of LINC00673 in cervical cancer cells was analyzed using in vitro and in vivo experiments.RESULTS:Our results revealed that serum LINC00673 levels were highest in cervical cancer patients, followed by patients with CIN and healthy controls. In vitro experiments demonstrated that overexpression of LINC00673 enhanced the proliferation and cell cycle progression of HeLa and SiHa cells. In vivo experiments showed that the tumor weight and volume of nude mice subcutaneously injected with LINC00673-overexpressing HeLa cells were larger than those of nude mice injected with control cells (P < 0.05). Western blotting showed that cell cycle-related proteins cyclin A2 and cyclin E and interstitial-associated proteins Snail and N-cadherin were upregulated and p53 signaling pathway-related proteins were downregulated in LINC00673-overexpressing HeLa and SiHa cells.CONCLUSION:LINC00673 plays an important role in the development of cervical cancer and may serve as a new therapeutic target for cervical cancer.

长非编码RNA SLC25A25-AS1在结直肠癌的发展中具有肿瘤抑制作用,然而,其在宫颈癌中作用机制有待深入研究.本文研究了宫颈癌和宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)病人血清中SLC25A25-AS1的异常表达,并探讨了SLC25A25-AS1在宫颈癌发展中可能的作用机制.采用RT-qPCR检测正常对照组,宫颈癌病人和CIN病人血清中SLC25A25-AS1的表达水平.在体外和体内研究中分析了SLC25A25-AS1在宫颈癌系HeLa细胞中的重要作用.与健康对照组相比,宫颈癌病人血清中的SLC25A25-AS1含量降低.在体外,SLC25A25-AS1的过表达抑制HeLa细胞的增殖、迁移和侵袭.体内裸鼠成瘤实验表明,注射过SLC25A25-AS1过表达的HeLa细胞的裸鼠皮下肿瘤的重量和体积明显小于注射对照细胞的裸鼠(P＜0.05).SLC25A25-AS1在宫颈癌的发生发展中可能发挥重要作用,有望成为宫颈癌的新治疗靶点.

The expression of RNA-binding proteins (RBPs) is dysregulated in colorectal cancer (CRC) and in other types of cancer. Among the RBPs, the insulin-like growth factor-2 messenger RNA binding protein (IGF2BP1-3) family is involved in the development of the colon and the progression of CRC. However, the regulation of mRNA fate by IGF2BP3 in CRC remains less well understood. Here, we show that IGF2BP3 interacts with ELAVL1 to coregulate a cohort of genes involved in the cell cycle and cell proliferation. Mechanistically, recognition of these mRNAs by the IGF2BP3/ELAVL1 complex leads to prolonged half-lives of the mRNA molecules and increased expression of the target genes, thereby driving CRC cell proliferation. Interestingly, knockdown of either IGF2BP3 or ELAVL1 impairs the IGF2BP3/ELAVL1 complex-enhanced mRNA stability, suggesting a functional interdependency between IGF2BP3 and ELAVL1 in CRC. Our findings reveal the molecular mechanism by which IGF2BP3 regulates mRNA stability and identify the cooperativity of the IGF2BP3/ELAVL1 complex as a novel therapeutic target in CRC.

Background We screened long non-coding RNAs (lncRNAs) specifically expressed in the serum of cervical squamous carcinoma (CESC) patient samples and investigated the role of these specific lncRNAs in the diagnosis of CESC and cervical intraepithelial neoplasia (CIN). Methods The expression levels of the lncRNAs CCAT2, LINC01133, and LINC00511 in the serum of normal controls and patient with CESC and CIN were measured using reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Next, we analyzed the correlation between the serum lncRNAs levels and the clinical characteristics of CESC. Thereafter, we estimated their combined diagnostic value by receiver operating characteristic (ROC) curve analysis. Results The results showed that CCAT2, LINC01133, and LINC00511 were highly expressed in the serum of patients with CESC. When these lncRNAs and squamous cell carcinoma (SCC) antigen were combined, the area under the ROC curve (AUC) value reached 0.94. We also found that the AUC value of the diagnostic model combining CCAT2 and LINC01133 reached 0.894. Conclusion The serum lncRNAs (CCAT2, LINC01133, and LINC00511) and SCC may be new non-invasive biomarkers for the diagnosis of CESC.

Normal hematopoiesis can be disrupted by the leukemic bone marrow microenvironment, which leads to cytopenia-associated symptoms including anemia, hemorrhage and infection. Thrombocytopenia is a major and sometimes fatal complication in patients with acute leukemia. However, the mechanisms underlying defective thrombopoiesis in leukemia have not been fully elucidated. In the steady state, platelets are continuously produced by megakaryocytes. Using an MLL-AF9-induced acute myeloid leukemia mouse model, we demonstrated a preserved number and proportion of megakaryocyte-primed hematopoietic stem cell subsets, but weakened megakaryocytic differentiation via both canonical and non-canonical routes. This primarily accounted for the dramatic reduction of megakaryocytic progenitors observed in acute myeloid leukemia bone marrow and a severe disruption of the maturation of megakaryocytes. Additionally, we discovered overproduction of interleukin-4 from bone marrow endothelial cells in acute myeloid leukemia and observed inhibitory effects of interleukin-4 throughout the process of megakaryopoiesis in vivo. Furthermore, we observed that inhibition of interleukin-4 in combination with induction chemotherapy not only promoted recovery of platelet counts, but also prolonged the duration of remission in our acute myeloid leukemia mouse model. Our study elucidates a new link between interleukin-4 signaling and defective megakaryopoiesis in acute myeloid leukemia bone marrow, thereby offering a potential therapeutic target in acute myeloid leukemia.

MiR-494 plays an important role in several types of human cancers, including non-small cell lung cancer (NSCLC). Although the role of miR-494 has been investigated in several studies, the expression profile and underlying mechanism are still poorly understood. In this study, we found that overexpression of miR-494 promoted the proliferation and colony formation of NSCLC cells and reduced their sensitivity to cisplatin-induced apoptosis. By using microarray and Dual luciferase reporter assays, we further showed that caspase-2 (CASP2) is a functional target of miR-494, and the expression of CASP2 is inversely associated with miR-494 in vitro. In addition, miR-494 promoted the proliferation and colony formation of NSCLC cells and reduced their sensitivity to cisplatin-induced apoptosis by targeting CASP2. Therefore, our results suggest that miR-494 plays an oncomiR role in NSCLC cells and may be a candidate biomarker for malignant transformation and a therapeutic target of NSCLC.

Abstract Background Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). Results We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. Conclusions These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

OBJECTIVE To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). METHODS Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. RESULTS In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G2/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. CONCLUSION The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.

Background: Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. Material/Methods: We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. Results: Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. Conclusions: These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells.

AIMS:Increasing evidence suggests that long noncoding RNAs act as critical regulators in various malignancies. Small nucleolar RNA host gene 6 (SNHG6) plays a role in the progression of human cancers. The present study aims to investigate the molecular mechanism through which SNHG6 promotes the development of gastric cancer (GC).MAIN METHODS:The expression level of SNHG6 in human serum was examined using reverse transcription and quantitative polymerase chain reaction (qRT-PCR). RNA- fluorescence in situ hybridization (FISH) and Cell nucleus/cytoplasm fraction isolation assay were used to detect the cellular distribution of SNHG6. Senescence-associated β-galactosidase (SA-β-gal) activity assay was performed to detect cell senescence. BALB/c male nude mice were used to establish the xenograft model.KEY FINDINGS:We found that SNHG6 was up-regulated in human GC tissues and serum. Knockdown of SNHG6 inhibited GC cell proliferation, induced cellular senescence, and reduced xenograft tumor growth in BALB/c nude mice. Knockdown of SNHG6 stimulated p21 expression and the tumor-suppressive effect of SNHG6 in GC cells was dependent on p21. Furthermore, the activation of the c-Jun N-terminal kinase (JNK) pathway and the decrease in Enhancer of Zeste Homolog 2 (EZH2) expression levels represented two mutually independent mechanisms by which SNHG6 knockdown resulted in the upregulation of p21.SIGNIFICANCE:Our findings show that SNHG6 knockdown inhibits GC development by upregulating p21; this effect is dependent on the activation of the JNK pathway and suppression of EZH2 expression. This study indicates that SNHG6 plays an important role in GC progression via the regulation of 21.

BACKGROUND The aim of this study was to identify a panel of serum noncoding RNAs (ncRNAs) as potential diagnostic and prognostic biomarkers for breast cancer. MATERIAL AND METHODS Patients with breast cancer (n=30), and normal controls (n=30) were included in the 'training set.' A 'validation set' included cases of breast cancer (n=128) and controls (n=77). All cases provided blood samples for serum analysis. All cases of breast cancer were confirmed histologically and were staged. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expression of 11 candidate ncRNAs, including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), in the serum. The expression of the panel of ncRNAs was further analyzed following surgery or chemotherapy. RESULTS The four ncRNAs identified in the serum of patients with breast cancer included let-7a, miR-155, miR-574-5p, and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Analysis based on the risk score showed that the panel of these four ncRNAs could effectively distinguish between patients with breast cancer and the control group. For the training set and the validation set, analysis of the receiver-operating characteristic (ROC) curve showed that the areas under the curve (AUCs) were 0.960 and 0.968, respectively. Also, the serum expression levels of the four ncRNAs differed in the pre-treatment and the post-treatment patients with breast cancer, with levels of miR-155 showing a significant decrease following chemotherapy. CONCLUSIONS A panel of serum ncRNAs, including let-7a, miR-155, miR-574-5p, and MALAT1, was shown to be present in patients with breast cancer.