Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno‐associated virus (AAV)‐mediated OTOF gene replacement therapy is extensively validated in Otof‐deficient mice. However, the clinical safety and efficacy of AAV‐OTOF is not reported. Here, AAV‐OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non‐human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV‐OTOF is delivered into one cochlea of a 5‐year‐old deaf patient and into the bilateral cochleae of an 8‐year‐old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure‐tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5‐year‐old patient can be restored to the normal range at 1 month after AAV‐OTOF injection, while the 8‐year‐old patient can hear the conversational sounds. Most importantly, the 5‐year‐old patient can hear and recognize speech only through the AAV‐OTOF‐injected ear. This study is the first to demonstrate the safety and efficacy of AAV‐OTOF in patients, expands and optimizes current OTOF‐related gene therapy and provides valuable information for further application of gene therapies for deafness.

Background: N6-methyladenosine (m6A) RNA methylation and its methyltransferase METTL3 have been widely reported to be involved in different cancers by regulating RNA metabolism and function. Here, we aimed to explore the biological function and clinical significance of m6A modification and METTL3 in head and neck squamous cell carcinoma (HNSCC). Methods: The prognostic value of METTL3 expression was evaluated using tissue microarray and immunohistochemical staining analyses in a human HNSCC cohort. The biological role and mechanism of METTL3 in HNSCC tumour growth, metastasis and angiogenesis were determined in vitro and in vivo. Results: M6A levels and METTL3 expressions in HNSCC tissues were significantly increased compared with paired adjacent tissues. Meanwhile, METTL3 was an independent risk factor for the prognosis of HNSCC patients. Moreover, METTL3 overexpression promoted HNSCC cell proliferation, migration, invasion, and angiogenesis, while knockdown of METTL3 had an opposite effect in vivo and in vitro. Mechanistically, METTL3 enhanced the m6A modification of CDC25B mRNA, which maintained its stability and upregulated its expression, thereby activating G2/M phase of cell cycle and leading to HNSCC malignant progression. Conclusions: METTL3 may be a potential prognostic biomarker and therapeutic target for HNSCC.

Cingulin (CGN) is a cytoskeleton‐associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi‐generation family presenting with autosomal dominant non‐syndromic hearing loss (ADNSHL) that co‐segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease‐causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell‐specific Cgn knockout leads to high‐frequency hearing loss. Importantly, Cgn mutation knockin mice display noise‐sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.

Objective: Over the past decade, heat shock protein 90 (HSP90) inhibitors have emerged as promising anticancer drugs in solid and hematological malignancies. Flavokawain C (FKC) is a naturally occurring chalcone that has been found to exert considerable anti-tumor efficacy by targeting multiple molecular pathways. However, the efficacy of FKC has not been studied in nasopharyngeal carcinoma (NPC). Metabolic abnormalities and uncontrolled angiogenesis are two important features of malignant tumors, and the occurrence of these two events may involve the regulation of HSP90B1. Therefore, this study aimed to explore the effects of FKC on NPC proliferation, glycolysis, and angiogenesis by regulating HSP90B1 and the underlying molecular regulatory mechanisms. Methods: HSP90B1 expression was analyzed in NPC tissues and its relationship with patient’s prognosis was further identified. Afterward, the effects of HSP90B1 on proliferation, apoptosis, glycolysis, and angiogenesis in NPC were studied by loss-of-function assays. Next, the interaction of FKC, HSP90B1, and epidermal growth factor receptor (EGFR) was evaluated. Then, in vitro experiments were designed to analyze the effect of FKC treatment on NPC cells. Finally, in vivo experiments were allowed to investigate whether FKC treatment regulates proliferation, glycolysis, and angiogenesis of NPC cells by HSP90B1/EGFR pathway. Results: HSP90B1 was highly expressed in NPC tissues and was identified as a poor prognostic factor in NPC. At the same time, knockdown of HSP90B1 can inhibit the proliferation of NPC cells, trigger apoptosis, and reduce glycolysis and angiogenesis. Mechanistically, FKC affects downstream EGFR phosphorylation by regulating HSP90B1, thereby regulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. FKC treatment inhibited the proliferation, glycolysis, and angiogenesis of NPC cells, which was reversed by introducing overexpression of HSP90B1. In addition, FKC can affect NPC tumor growth and metastasis in vivo by regulating the HSP90B1/EGFR pathway. Conclusion: Collectively, FKC inhibits glucose metabolism and tumor angiogenesis in NPC by targeting the HSP90B1/EGFR/PI3K/Akt/mTOR signaling axis.

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α‐ketoglutarate (α‐KG) and nicotinamide adenine dinucleotide (NAD⁺). Reduced expression of NAD⁺ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α‐KG and NAD⁺ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.