AAV-ie-Tnfaip8l2的包装及其对Tnfaip8l2^-/-小鼠听觉功能的改善效果

Packaging of AAV-ie-Tnfaip8l2 and its effect on improving auditory function in Tnfaip8l2^-/- mice

摘要

目的 腺相关病毒(adeno-associated virus, AAV)是基因治疗的重要载体。本研究旨在包装AAV-ie-Tnfaip8l2病毒,系统评估能否以AAV为载体实现Tnfaip8l2蛋白在耳蜗的原位过表达,并探索其在基因敲除小鼠模型内耳组织中的转导效率及对该基因敲除小鼠的听觉功能治疗效果。方法 采用分子克隆技术构建重组表达载体AAV-ie-Tnfaip8l2。将AAV-ie衣壳质粒及辅助质粒共转染至293T细胞中,进行病毒颗粒的组装和复制。通过超速离心和层析纯化获得高纯度、高滴度的重组腺相关病毒制剂。运用显微注射技术,经圆窗膜对Tnfaip8l2^-/-新生鼠进行圆窗注射AAV-ie-Tnfaip8l2。待Tnfaip8l2^-/-小鼠生长至35 d龄(典型听力损失显现期)时,通过听力检测、扫描电镜、免疫荧光等方法评估疗效。结果 高滴度的AAV-ie-Tnfaip8l2病毒可成功包装用于耳蜗圆窗注射,以实现Tnfaip8l2基因在Tnfaip8l2^-/-小鼠耳蜗原位高效过表达。听力学检测显示,与对照组相比,AAV-ie-Tnfaip8l2回补组小鼠Click、短纯音(4 k、8 k、12 k、16 k、24 k、32 k)以及DPOAE阈值降低(P<0.05,n=3);扫描电镜显示外毛细胞静纤毛束排列紊乱现象得到了明显的改善。结论 本研究证实AAV-ie-Tnfaip8l2可通过圆窗注射实现Tnfaip8l2基因在小鼠耳蜗原位的高效表达,部分挽救Tnfaip8l2^-/-小鼠的听力功能,为老年性耳聋的治疗方案提供新的实验依据。

Abstract

Objective Adeno-associated virus (AAV) serves as a critical vector for gene therapy. This study aimed to package AAV-ie-Tnfaip8l2 virus, systematically evaluate whether AAV could be used as a vector to achieve in situ overexpression of Tnfaip8l2 protein in the cochlea, and explore its transduction efficiency in the inner ear tissues of gene knockout mouse models and therapeutic effects on auditory function. Methods The recombinant expression vector AAV-ie-Tnfaip8l2 was constructed using molecular cloning techniques. AAV-ie capsid plasmids and helper plasmids were co-transfected into 293T cells to complete the assembly and replication of viral particles. High-purity, high-titer recombinant AAV preparations were obtained via ultracentrifugation and chromatography purification. Using microinjection technology, AAV-ie-Tnfaip8l2 was injected into the round window of newborn Tnfaip8l2^-/- mice through the round window membrane. When the Tnfaip8l2^-/- mice reached 35 days of age (the typical period when hearing loss becomes apparent), the therapeutic effects were evaluated using methods such as audiological tests, scanning electron microscopy, and immunofluorescence. Results High-titer AAV-ie-Tnfaip8l2 virus could be successfully packaged for cochlear round window injection, enabling efficient in situ overexpression of the Tnfaip8l2 in the cochlea of Tnfaip8l2^-/- mice. Audiological function tests showed that compared with the control groups, the click, tone burst (4 k, 8 k, 12 k, 16 k, 24 k, 32 k), and DPOAE thresholds in the AAV-ie-Tnfaip8l2 replenishment group mice were significantly reduced (P<0.05, n=3). Scanning electron microscopy revealed that the disordered arrangement of stereociliary bundles in outer hair cells was significantly improved. Conclusion This study confirmed that AAV-ie-Tnfaip8l2 can achieve efficient in situ expression of the Tnfaip8l2 in the mouse cochlea via round window injection, partially rescuing the hearing function of Tnfaip8l2^-/- mice. These findings provide new experimental evidence for therapeutic strategies for age-related hearing loss.

关键词

腺相关病毒 / 听力损失 / TNFAIP8L2 / 基因功能补偿

Key words

Adeno-associated virus / Hearing loss / TNFAIP8L2 / Gene function compensation

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基金资助

国家自然科学基金项目(82201296)