RNA base editing therapy cures hearing loss induced by OTOF gene mutation.

Otoferlin (OTOF) gene mutations represent the primary cause of hearing impairment and deafness in auditory neuropathy. The c.2485C>T (p. Q829X) mutation variant is responsible for approximately 3% of recessive prelingual deafness cases within the Spanish population. Previous studies have used two recombinant AAV vectors to overexpress OTOF, albeit with limited efficacy. In this study, we introduce an enhanced mini-dCas13X RNA base editor (emxABE) delivered via an AAV9 variant, achieving nearly 100% transfection efficiency in inner hair cells. This approach is aimed at treating OTOFQ829X, resulting in an approximately 80% adenosine-to-inosine conversion efficiency in humanized OtofQ829X/Q829X mice. Following a single scala media injection of emxABE targeting OTOFQ829X (emxABE-T) administered during the postnatal day 0-3 period in OtofQ829X/Q829X mice, we observed OTOF expression restoration in nearly 100% of inner hair cells. Moreover, auditory function was significantly improved, reaching similar levels as in wild-type mice. This enhancement persisted for at least 7 months. We also investigated P5-P7 and P30 OtofQ829X/Q829X mice, achieving auditory function restoration through round window injection of emxABE-T. These findings not only highlight an effective therapeutic strategy for potentially addressing OTOFQ829X-induced hearing loss but also underscore emxABE as a versatile toolkit for treating other monogenic diseases characterized by premature termination codons.

Establishment of a Seven-Gene Signature Associated with CD8+ T Cells through the Utilization of Both Single-Cell and Bulk RNA-Sequencing Techniques in Clear Cell Renal Cell Carcinoma.

Tumor immune microenvironment constituents, such as CD8+ T cells, have emerged as crucial focal points for cancer immunotherapy. Given the absence of reliable biomarkers for clear cell renal cell carcinoma (ccRCC), we aimed to ascertain a molecular signature that could potentially be linked to CD8+ T cells. The differentially expressed genes (DEGs) linked to CD8+ T cells were identified through an analysis of single-cell RNA sequencing (scRNA-seq) data obtained from the Gene Expression Omnibus (GEO) database. Subsequently, immune-associated genes were obtained from the InnateDB and ImmPort datasets and were cross-referenced with CD8+ T-cell-associated DEGs to generate a series of DEGs linked to immune response and CD8+ T cells. Patients with ccRCC from the Cancer Genome Atlas (TCGA) were randomly allocated into testing and training groups. A gene signature was established by conducting LASSO-Cox analysis and subsequently confirmed using both the testing and complete groups. The efficacy of this signature in evaluating immunotherapy response was assessed on the IMvigor210 cohort. Finally, we employed various techniques, including CIBERSORT, ESTIMATE, ssGSEA, and qRT-PCR, to examine the immunological characteristics, drug responses, and expression of the signature genes in ccRCC. Our findings revealed 206 DEGs linked to immune response and CD8+ T cells, among which 65 genes were correlated with overall survival (OS) in ccRCC. A risk assessment was created utilizing a set of seven genes: RARRES2, SOCS3, TNFSF14, XCL1, GRN, CLDN4, and RBP7. The group with a lower risk showed increased expression of CD274 (PD-L1), suggesting a more favorable response to anti-PD-L1 treatment. The seven-gene signature demonstrated accurate prognostic prediction for ccRCC and holds potential as a clinical reference for treatment decisions.

Functional analysis of the heterotrimeric NF-Y transcription factor complex in cassava disease resistance.

BACKGROUND AND AIMS: The nuclear factor Y (NF-Y) transcription factor complex is important in plant growth, development and stress response. Information regarding this transcription factor complex is limited in cassava (Manihot esculenta). In this study, 15 MeNF-YAs, 21 MeNF-YBs and 15 MeNF-YCs were comprehensively characterized during plant defence. METHODS: Gene expression in MeNF-Ys was examined during interaction with the bacterial pathogen Xanthomonas axonopodis pv. manihotis (Xam). The yeast two-hybrid system was employed to investigate protein-protein interactions in the heterotrimeric NF-Y transcription factor complex. The in vivo roles of MeNF-Ys were revealed by virus-induced gene silencing (VIGS) in cassava. KEY RESULTS: The regulation of MeNF-Ys in response to Xam indicated their possible roles in response to cassava bacterial blight. Protein-protein interaction assays identified the heterotrimeric NF-Y transcription factor complex (MeNF-YA1/3, MeNF-YB11/16 and MeNF-YC11/12). Moreover, the members of the heterotrimeric NF-Y transcription factor complex were located in the cell nucleus and conferred transcriptional activation activity to the CCAAT motif. Notably, the heterotrimeric NF-Y transcription factor complex positively regulated plant disease resistance to Xam, confirmed by a disease phenotype in overexpressing plants in Nicotiana benthamiana and VIGS in cassava. Consistently, the heterotrimeric NF-Y transcription factor complex positively regulated the expression of pathogenesis-related genes (MePRs). CONCLUSIONS: The NF-Y transcription factor complex (MeNF-YA1/3, MeNF-YB11/16 and MeNF-YC11/12) characterized here was shown to play a role in transcriptional activation of MePR promoters, contributing to the plant defence response in cassava.

Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava.

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and ß-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

Molecular identification of GAPDHs in cassava highlights the antagonism of MeGAPCs and MeATG8s in plant disease resistance against cassava bacterial blight.

KEY MESSAGE: MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.