Abstract Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1 , in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity, with Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.
Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a "multiple vectors in one AAV" strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof -/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.
Abstract Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss 9 (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual AAV1 carrying human OTOF coding sequences with the expression driven by the hair cell-specific promoter Myo15 , AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models by pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof −/− mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea although it was detected in other organs such as the central nervous system and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60-94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.
Introduction This study examined the impact of biochar application on agricultural productivity and greenhouse gas emissions in irrigated regions of northern Xinjiang. The objective of this study was to assess the impact of nitrogen fertilizer and biochar levels on soil respiration rate, enzyme activity, and spring wheat yield. Materials and methods The experiment employed a randomized block design comprising two nitrogen fertilizer levels (N1: 300 kg·hm -2 and N2: 255 kg·hm -2 ) and four biochar levels (B0: 0 kg·hm -2 , B1: 10×10 3 kg·hm -2 , B2: 20×10 3 kg·hm -2 , and B3: 30×10 3 kg·hm -2 ). This resulted in eight groups (N1B0, N1B1, N1B2, N1B3, N2B0, N2B1, N2B2, and N2B3), each replicated three times. Results and discussion The findings indicated that the N2B2 group exhibited a reduction in soil CO 2 emissions, with a cumulative decrease of 4.42% in CO 2 emissions compared to the N2B0 control. The application of biochar and/or nitrogen fertilizer, particularly in combination, was observed to increase soil urease, sucrase, and catalase activities. The N2B2 group exhibited a spring wheat yield of 8301.35 kg·hm -2 , representing a 22.1% increase over the N1B0 group. This improvement was attributed to the capacity of biochar to regulate soil water content variability, stabilize soil aggregate composition, mitigate organic carbon mineralization, and reduce farmland carbon emissions. Furthermore, biochar’s nitrogen fixation provided essential nutrients for soil microorganisms, thereby enhancing enzymatic reactions and promoting crop growth. Conclusion In conclusion, the N2B2 regime was determined to be the optimal approach for spring wheat cultivation in irrigated regions of northern Xinjiang, resulting in enhanced crop productivity and the mitigation of carbon emissions. Nevertheless, further investigation of its long-term impact on farmland is recommended.
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