Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of ...human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.
The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell ...lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing.
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Numerous studies have shown Kinesin family member 20A (KIF20A) may play a critical role in the development and progression of cancer. However, the clinical value of KIF20A in nasopharyngeal carcinoma ...(NPC) is unknown. Here, we investigated the expression pattern of KIF20A in NPC and its correlation with clinicopathological features of patients.
Real-time PCR and Western blotting were used to quantify KIF20A expression in NPC cell lines and clinical specimens compared with normal controls. KIF20A protein expression was also examined in archived paraffin embedded tumor samples from 105 patients with pathologically confirmed NPC by immunohistochemistry (IHC). Statistical analyses were applied to assess the associations between KIF20A expression and the clinicopathological features and survival outcomes. Effects on migration and invasion were assessed by wound healing and transwell invasion assays after KIF20A silencing.
KIF20A was significantly overexpressed at both the mRNA and protein levels in NPC cell lines and human tumor tissues. 45/105 (42.9%) of NPC specimens expressed high levels of KIF20A among the KIF20A detectable cases. Statistical analysis revealed that high KIF20A expression was significantly associated with gender (P = 0.046), clinical stage (P<0.001), T category (P = 0.022), N category (P<0.001), distant metastasis (P = 0.001) and vital status (P = 0.001). Moreover, Higher KIF20A expression patients had shorter overall survival (OS) and progression-free survival (PFS) (P = 0.001 and P = 0.001; log-rank test). In multivariate analysis, KIF20A was an independent prognostic factor for OS and PFS in the entire cohort (P = 0.033, P = 0.008). Knock down of KIF20A expression significantly suppressed NPC cell's migration and invasion.
KIF20A is overexpressed and may serve as an independent prognostic biomarker in NPC. Targeting KIF20A reduces migration and invasion of NPC cells.
Previous studies demonstrated that the radiation therapy, image technology, and the application of chemotherapy have developed in the last 2 decades. This study explored the survival trends and ...treatment failure patterns of patients with nonmetastatic nasopharyngeal carcinoma (NPC) treated with radiation therapy. Furthermore, we evaluated the survival benefit brought by the development of radiation therapy, image technology, and chemotherapy based on a large cohort from 1990 to 2012.
Data from 20,305 patients with nonmetastatic NPC treated between 1990 and 2012 were analyzed. Patients were divided into 4 calendar periods (1990-1996, 1997-2002, 2003-2007, and 2008-2012). Overall survival (OS) was the primary endpoint.
Magnetic resonance imaging has replaced computed tomography as the most important imaging technique since 2003. Conventional 2-dimensional radiation therapy, which was the main radiation therapy technique in our institution before 2008, was replaced by intensity modulated radiation therapy later. An increasing number of patients have undergone chemotherapy since 2003. The 5-year OS across the 4 calendar periods increased at each TNM stage with progression-free survival (PFS) and locoregional relapse-free survival (LRFS) showing a similar trend, whereas distant metastasis-free survival showed small differences. Multivariate analyses showed that the application of intensity modulated radiation therapy and magnetic resonance imaging were independent protective factors in OS, PFS, LRFS, and distant metastasis-free survival. Chemotherapy benefited patients in OS, PFS, and LRFS. The main pattern of treatment failure shifted from recurrence to distant metastasis.
The development of radiation therapy, image technology, and chemotherapy increased survival rates among patients with NPC because of excellent locoregional control. Distant failure has become the greatest challenge for NPC treatment.
Patients with N2–3 nasopharyngeal carcinoma have a high risk of treatment being unsuccessful despite the current practice of using a concurrent adjuvant cisplatin–fluorouracil regimen. We aimed to ...compare the efficacy and safety of concurrent adjuvant cisplatin–gemcitabine with cisplatin–fluorouracil in N2–3 nasopharyngeal carcinoma.
We conducted an open-label, randomised, controlled, phase 3 trial at four cancer centres in China. Eligible patients were aged 18–65 years with untreated, non-keratinising, stage T1–4 N2–3 M0 nasopharyngeal carcinoma, an Eastern Cooperative Oncology Group performance status score of 0–1, and adequate bone marrow, liver, and renal function. Eligible patients were randomly assigned (1:1) to receive concurrent cisplatin (100 mg/m2 intravenously) on days 1, 22, and 43 of intensity-modulated radiotherapy followed by either gemcitabine (1 g/m2 intravenously on days 1 and 8) and cisplatin (80 mg/m2 intravenously for 4 h on day 1) once every 3 weeks or fluorouracil (4 g/m2 in continuous intravenous infusion for 96 h) and cisplatin (80 mg/m2 intravenously for 4 h on day 1) once every 4 weeks, for three cycles. Randomisation was done using a computer-generated random number code with a block size of six, stratified by treatment centre and nodal category. The primary endpoint was 3-year progression-free survival in the intention-to-treat population (ie, all patients randomly assigned to treatment). Safety was assessed in all participants who received at least one dose of chemoradiotherapy. This study was registered at ClinicalTrials.gov, NCT03321539, and patients are currently under follow-up.
From Oct 30, 2017, to July 9, 2020, 240 patients (median age 44 years IQR 36–52; 175 73% male and 65 27% female) were randomly assigned to the cisplatin–fluorouracil group (n=120) or cisplatin–gemcitabine group (n=120). As of data cutoff (Dec 25, 2022), median follow-up was 40 months (IQR 32–48). 3-year progression-free survival was 83·9% (95% CI 75·9–89·4; 19 disease progressions and 11 deaths) in the cisplatin–gemcitabine group and 71·5% (62·5–78·7; 34 disease progressions and seven deaths) in the cisplatin–fluorouracil group (stratified hazard ratio 0·54 95% CI 0·32–0·93; log rank p=0·023). The most common grade 3 or worse adverse events that occurred during treatment were leukopenia (61 52% of 117 in the cisplatin–gemcitabine group vs 34 29% of 116 in the cisplatin–fluorouracil group; p=0·00039), neutropenia (37 32% vs 19 16%; p=0·010), and mucositis (27 23% vs 32 28%; p=0·43). The most common grade 3 or worse late adverse event (occurring from 3 months after completion of radiotherapy) was auditory or hearing loss (six 5% vs ten 9%). One (1%) patient in the cisplatin–gemcitabine group died due to treatment-related complications (septic shock caused by neutropenic infection). No patients in the cisplatin–fluorouracil group had treatment-related deaths.
Our findings suggest that concurrent adjuvant cisplatin–gemcitabine could be used as an adjuvant therapy in the treatment of patients with N2–3 nasopharyngeal carcinoma, although long-term follow-up is required to confirm the optimal therapeutic ratio.
National Key Research and Development Program of China, National Natural Science Foundation of China, Guangdong Major Project of Basic and Applied Basic Research, Sci-Tech Project Foundation of Guangzhou City, Sun Yat-sen University Clinical Research 5010 Program, Innovative Research Team of High-level Local Universities in Shanghai, Natural Science Foundation of Guangdong Province for Distinguished Young Scholar, Natural Science Foundation of Guangdong Province, Postdoctoral Innovative Talent Support Program, Pearl River S&T Nova Program of Guangzhou, Planned Science and Technology Project of Guangdong Province, Key Youth Teacher Cultivating Program of Sun Yat-sen University, the Rural Science and Technology Commissioner Program of Guangdong Province, and Fundamental Research Funds for the Central Universities.
Objectives/Hypothesis
The routine practices of examining submucosal lesions are not suitable for deep lesions. Therefore, we evaluated the efficacy of non‐real‐time image‐guided transnasal endoscopic ...fine‐needle aspiration biopsy (FNAB) in diagnosing nasopharyngeal carcinoma (NPC) with submucosal lesions.
Study Design
The effectiveness evaluation of diagnostic methods.
Methods
Fifty suspected NPC patients who failed in conventional biopsies were enrolled in this study. The efficacy, maneuverability, and safety of FNAB in diagnosing these intractable cases were evaluated.
Results
The definitive diagnostic results of these 50 patients were NPC (34/50, 68.0%), nasopharyngeal necrosis (1/50, 2.0%), nasopharyngeal mucositis (12/50, 24.0%), and other cancers (3/50, 6.0%), respectively. The results of the diagnostic efficacy of FNAB were sensitivity, 89.2%; specificity, 100.0%; positive predictive value, 100.0%; negative predictive value, 76.5%; and accuracy, 92.0%, respectively. The area under the receiver operating characteristic curves was 0.946 (95% confidence interval = 0.884–1.00, P < .001). No severe complications occurred after FNAB.
Conclusions
FNAB can improve the diagnostic efficiency of NPC occurring in the submucosal space. It can be an additional option for routine nasopharyngeal biopsy and is worthy of clinical application.
Level of Evidence
4 Laryngoscope, 131:1798–1804, 2021
Early diagnosis is critical to reduce the mortality caused by nasopharyngeal carcinoma (NPC). MicroRNAs (miRNAs) are dysregulated and play important roles in carcinogenesis. Therefore, this study ...aimed to identify diagnostically relevant circulating miRNA signatures in patients with NPC.
Total RNA was extracted from whole blood samples obtained from 120 patients with NPC, 30 patients with head-neck tumors (HNT), and 30 healthy subjects (HSs), and examined by using a custom microarray. The expression levels of four miRNAs identified by using the microarray were validated with quantitative real-time reverse transcription polymerase chain reaction. The 120 patients with NPC and 30 HSs were randomly assigned to training group-1 and validation group-1, respectively. By using significance analysis of microarray (SAM), the specific miRNA expression profiles in whole blood from patients with NPC are obtained. By using lasso regression and adaptive boosting, a diagnostic signature was identified in training group-1, and its accuracy was verified in validation group-1. By using the same methods, another signature to distinguish patients with NPC from those with HNT and HSs was identified in training group-2 and confirmed in validation group-2.
There were 117 differentially expressed miRNAs (upregulated and downregulated fold change ≥ 1.5) between the patients with NPC and HSs, among which an 8-miRNA signature was identified with 96.43% sensitivity and 100% specificity area under the curve (AUC) = 0.995 to diagnose NPC in training group-1 and 86.11% sensitivity and 88.89% specificity (AUC = 0.941) in validation group-1. Compared with traditional Epstein-Barr virus (EBV) seromarkers, this signature was more specific for NPC. Furthermore, a 16-miRNA signature to differentiate NPC from HNT and HS (HNT-HS) was established from 164 differentially expressed miRNAs, which diagnosed NPC and HNT-HS with 100% accuracy (AUC = 1.000) in training group-2 and 87.04% (AUC = 0.924) in validation group-2.
The present study identified two miRNA signatures for the highly accurate diagnosis and differential diagnosis of patients with NPC from HSs and patients with HNT. The identified miRNAs might represent novel serological biomarkers and potential therapeutic targets for NPC.
Immunotherapy combined with antiangiogenic targeted therapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for refractory recurrent/metastatic ...nasopharyngeal carcinoma (RM-NPC). We conducted a phase 2 trial to evaluate the safety and activity of camrelizumab plus apatinib in platinum-resistant (cohort 1, NCT04547088) and PD-1 inhibitor resistant NPC (cohort 2, NCT04548271). Here we report on the primary outcome of objective response rate (ORR) and secondary endpoints of safety, duration of response, disease control rate, progression-free survival, and overall survival. The primary endpoint of ORR was met for cohort 1 (65%, 95% CI, 49.6-80.4, n = 40) and cohort 2 (34.3%; 95% CI, 17.0-51.8, n = 32). Grade ≥ 3 treatment-related adverse events (TRAE) were reported in 47 (65.3%) of 72 patients. Results of our predefined exploratory investigation of predictive biomarkers show: B cell markers are the most differentially expressed genes in the tumors of responders versus non-responders in cohort 1 and that tertiary lymphoid structure is associated with higher ORR; Angiogenesis gene expression signatures are strongly associated with ORR in cohort 2. Camrelizumab plus apatinib combination effectiveness is associated with high expression of PD-L1, VEGF Receptor 2 and B-cell-related genes signatures. Camrelizumab plus apatinib shows promising efficacy with a measurable safety profile in RM-NPC patients.
Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barré syndrome
. While progress has been made in understanding the causal link between ZIKV infection and microcephaly
, the ...life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV
. Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.
•There is need to explore inexpensive and trustworthy biomarkers for poor prognosis.•We examined prognostic value of a pre-treatment peripheral blood signature.•NPC patients were distinguished by ...their peripheral blood signature scores.•Our peripheral blood signature-based nomogram offers individualised risk estimation.
We sought to determine the prognostic value of a pre-treatment peripheral blood signature and the peripheral blood signature-based nomogram for patients with non-metastatic nasopharyngeal carcinoma (NPC).
We retrospectively collected 21 peripheral blood indicators from patients with NPC between 2004 and 2015. Data were randomly divided into a training and a validation set (ratio: 6:4). The peripheral blood signature was constructed based on candidate biomarkers using the least absolute shrinkage and selection operator Cox regression model. Multivariable logistic regression was applied to identify the independent risk factors of overall survival to build the nomogram. The predictive value of the peripheral blood nomogram was evaluated using time-dependent area under the curve, decision curve analysis, and calibration curve.
In total, 6668 patients were enrolled with 4000 and 2668 in the training and validation cohorts, respectively. Four peripheral blood indicators, (white blood cell count, lymphocyte percentage, haemoglobin, and mean platelet volume), were included to construct the peripheral blood signature. Patients were divided into low- and high-risk groups using an optimal cut-off value of − 1.71142. Patients in the high-risk group had significantly lower overall, distant metastasis-free, and progression-free survival than patients in the low-risk group in both cohorts (P < 0.05). We constructed and validated a peripheral blood signature-based nomogram in combination with five vital clinical characteristics, (age, sex, tumour stage, nodal stage, and pre-treatment Epstein–Barr virus DNA), which showed favourable performance.
Patients with NPC with different outcomes could be distinguished based on their peripheral blood signature score; the proposed peripheral blood signature-based nomogram offers individualised risk estimation.
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