Seist Richard, Copeland Juwan S, Tao Litao, Groves Andrew K
Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Program in Genetics & Genomics, Baylor College of Medicine, Houston, TX 77030, USA.
Hear Res. 2025 Mar;458:109203. doi: 10.1016/j.heares.2025.109203. Epub 2025 Jan 28.
Achieving cell-specific gene expression is crucial in the design of safe and efficacious gene therapies for the treatment of sensorineural hearing loss. Although a variety of adeno-associated virus (AAV) serotypes have been used to deliver genes to inner ear hair cells, few serotypes currently allow specific targeting of supporting cells. We sought to specifically target supporting cells by combining an AAV serotype with high tropism for the inner ear with enhancer sequences from the supporting cell-specific gene Lunatic Fringe (Lfng). We identified three candidate Lfng enhancer sequences using bioinformatic analysis to identify accessible chromatin and histone marks associated with active transcription of the Lfng locus in supporting cells. Candidate Lfng enhancers or the ubiquitous CBh promoter driving an EGFP reporter gene were packaged into the AAV-ie capsid, and the virus was introduced into the inner ear of neonatal mice. AAV-CBh-EGFP transduced multiple sensory and non-sensory inner ear cell types, as well as cells in the brain. One of the three Lfng enhancers gave robust EGFP expression in border cells, inner phalangeal cells, pillar cells, and all three rows of Deiters' cells along the entire cochlear duct, as well as in vestibular organ supporting cells. Significantly, no fluorescently labeled cells were detected in the brains of mice injected with this virus. We further designed an AAV-Lfng-CreERT2 vector that drove strong recombination in Cre reporter mice supporting cells after tamoxifen treatment. Our results provide a tool to specifically target supporting cells of the juvenile and adult inner ear.
实现细胞特异性基因表达对于设计安全有效的基因疗法治疗感音神经性听力损失至关重要。尽管多种腺相关病毒（AAV）血清型已被用于将基因传递至内耳毛细胞，但目前很少有血清型能够特异性靶向支持细胞。我们试图通过将对内耳具有高嗜性的AAV血清型与支持细胞特异性基因Lunatic Fringe（Lfng）的增强子序列相结合，来特异性靶向支持细胞。我们使用生物信息学分析来鉴定与支持细胞中Lfng基因座的活跃转录相关的可及染色质和组蛋白标记，从而确定了三个候选Lfng增强子序列。将候选Lfng增强子或驱动EGFP报告基因的普遍存在的CBh启动子包装到AAV-ie衣壳中，并将病毒引入新生小鼠的内耳。AAV-CBh-EGFP转导了多种感觉和非感觉性内耳细胞类型以及脑中的细胞。三个Lfng增强子之一在边界细胞、内指细胞、柱细胞以及沿整个耳蜗管的所有三排Deiters细胞以及前庭器官支持细胞中均产生了强烈的EGFP表达。重要的是，在注射该病毒的小鼠脑中未检测到荧光标记的细胞。我们进一步设计了一种AAV-Lfng-CreERT2载体，该载体在他莫昔芬处理后在Cre报告基因小鼠的支持细胞中驱动了强烈的重组。我们的结果提供了一种特异性靶向幼年和成年内耳支持细胞 的工具。
