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

doi:10.6040/j.issn.1673-3770.0.2025.177

摘要/Abstract

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。

关键词: 腺相关病毒, 听力损失, TNFAIP8L2, 基因功能补偿

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 Tnfaip812^-/- 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.

Key words: Adeno-associated virus, Hearing loss, TNFAIP8L2, Gene function compensation

中图分类号:

R764

王敏,刘皓,姜露涵,谭立约,李文,付小龙. AAV-ie-Tnfaip8l2的包装及其对Tnfaip8l2^-/-小鼠听觉功能的改善效果[J]. 山东大学耳鼻喉眼学报, 2026, 40(3): 7-15.

WANG Min, LIU Hao, JIANG Luhan, TAN Liyue, LI Wen, FU Xiaolong. Packaging of AAV-ie-Tnfaip8l2 and its effect on improving auditory function in Tnfaip8l2^-/- mice[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2026, 40(3): 7-15.

参考文献

[1] Pupo A, Fernández A, Low SH, et al. AAV vectors: the Rubik's cube of human gene therapy[J]. Mol Ther, 2022, 30(12): 3515-3541. doi:10.1016/j.ymthe.2022.09.015
[2] Hu mL, Edwards TL, O’Hare F, et al. Gene therapy for inherited retinal diseases: progress and possibilities[J]. Clin Exp Optom, 2021, 104(4): 444-454. doi:10.1080/08164622.2021.1880863
[3] He XJ, Urip BA, Zhang ZJ, et al. Evolving AAV-delivered therapeutics towards ultimate cures[J]. J Mol Med, 2021, 99(5): 593-617. doi:10.1007/s00109-020-02034-2
[4] Lv J, Wang H, Cheng XT, et al. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial[J]. Lancet, 2024, 403(10441): 2317-2325. doi:10.1016/S0140-6736(23)02874-X
[5] Qi JY, Tan FZ, Zhang LY, et al. AAV-mediated gene therapy restores hearing in patients with DFNB9 deafness[J]. Adv Sci, 2024, 11(11): 2306788. doi:10.1002/advs.202306788
[6] Qi JY, Xu L, Zeng FG, et al. OTOF-related gene therapy: a new way but a long road ahead[J]. Lancet, 2025, 405(10481): 777-779. doi:10.1016/S0140-6736(25)00248-X
[7] Sun GQ, Zheng YD, Fu XL, et al. Single-cell transcriptomic atlas of mouse cochlear aging[J]. Protein Cell, 2023, 14(3): 180-201. doi:10.1093/procel/pwac058
[8] Eshel M, Milon B, Hertzano R, et al. The cells of the sensory epithelium, and not the stria vascularis, are the main cochlear cells related to the genetic pathogenesis of age-related hearing loss[J]. Am J Hum Genet, 2024, 111(3): 614-617. doi:10.1016/j.ajhg.2024.01.008
[9] Ege T, Tao LT, North BJ. The role of molecular and cellular aging pathways on age-related hearing loss[J]. Int J Mol Sci, 2024, 25(17): 9705. doi:10.3390/ijms25179705
[10] Fu XL, Sun XY, Zhang LQ, et al. Tuberous sclerosis complex-mediated mTORC1 overactivation promotes age-related hearing loss[J]. J Clin Invest, 2018, 128(11): 4938-4955. doi:10.1172/JCI98058
[11] Fu XL, Li PP, Zhang LQ, et al. Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss[J]. Proc Natl Acad Sci USA, 2022, 119(10): e2107357119. doi:10.1073/pnas.2107357119
[12] Li W, Li YL, Guan YT, et al. TNFAIP8L2/TIPE2 impairs autolysosome reformation via modulating the RAC1-MTORC1 axis[J]. Autophagy, 2021, 17(6): 1410-1425. doi:10.1080/15548627.2020.1761748
[13] Li W, Li Y, Wang M, et al. TNFAIP8L2 maintains hair cell function and regulates age-related hearing loss via mTORC1 signaling[J]. Mol Ther, 2025, 33(7): 3036-3055. doi:10.1016/j.ymthe.2025.03.046
[14] Sun HH, Gong SY, Carmody RJ, et al. TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis[J]. Cell, 2008, 133(3): 415-426. doi:10.1016/j.cell.2008.03.026
[15] Zhang LY, Chen X, Wang XL, et al. AAV-mediated gene cocktails enhance supporting cell reprogramming and hair cell regeneration[J]. Adv Sci, 2024, 11(29): 2304551. doi:10.1002/advs.202304551
[16] Andres-Mateos E, Landegger LD, Unzu C, et al. Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate[J]. Nat Commun, 2022, 13(1): 1359. doi:10.1038/s41467-022-28969-3
[17] Fu XL, Wan PF, Lu L, et al. Peroxisome deficiency in cochlear hair cells causes hearing loss by deregulating BK channels[J]. Adv Sci, 2023, 10(20): 2300402. doi:10.1002/advs.202300402
[18] Lee YY, Ha J, Kim YS, et al. Abnormal cholesterol metabolism and lysosomal dysfunction induce age-related hearing loss by inhibiting mTORC1-TFEB-dependent autophagy[J]. Int J Mol Sci, 2023, 24(24): 17513. doi:10.3390/ijms242417513
[19] Tan FZ, Chu CF, Qi JY, et al. AAV-ie enables safe and efficient gene transfer to inner ear cells[J]. Nat Commun, 2019, 10(1): 3733. doi:10.1038/s41467-019-11687-8
[20] Chen Y, Xu ZY, Sun HX, et al. Regulation of CD⁸⁺ T memory and exhaustion by the mTOR signals[J]. Cell Mol Immunol, 2023, 20(9): 1023-1039. doi:10.1038/s41423-023-01064-3
[21] Costa Verdera H, Kuranda K, Mingozzi F. AAV vector immunogenicity in humans: a long journey to successful gene transfer[J]. Mol Ther, 2020, 28(3): 723-746. doi:10.1016/j.ymthe.2019.12.010
[22] Ivanchenko MV, Hathaway DM, Mulhall EM, et al. PCDH15 dual-AAV gene therapy for deafness and blindness in Usher syndrome type 1F models[J]. J Clin Investig, 2024, 134(23): e177700. doi:10.1172/jci177700
[23] Mingozzi F, Meulenberg JJ, Hui DJ, et al. AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells [J]. Blood, 2009, 114(10):2077-2086.doi:10.1182/blood-2008-07-167510
[24] Hinderer C, Katz N, Buza EL, et al. Severe toxicity in nonhuman Primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN[J]. Hum Gene Ther, 2018, 29(3): 285-298. doi:10.1089/hum.2018.015
[25] Weber T. Anti-AAV antibodies in AAV gene therapy: current challenges and possible solutions[J]. Front Immunol, 2021, 12: 658399. doi:10.3389/fimmu.2021.658399
[26] Martins KM, Breton C, Zheng Q, et al. Prevalent and disseminated recombinant and wild-type adeno-associated virus integration in macaques and humans[J]. Hum Gene Ther, 2023: hum.2023.134. doi:10.1089/hum.2023.134
[27] Qi J, Fu X, Zhang L, et al. Current AAV-mediated gene therapy in sensorineural hearing loss [J]. Fundam Res, 2022, 5(1): 192-202.doi:10.1016/j.fmre.2022.08.015
[28] Lau SC, Grati M, Isgrig K, et al. Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B[J]. Mol Ther Methods Clin Dev, 2023, 30: 534-545. doi:10.1016/j.omtm.2023.08.012
[29] Liu SY, Song WQ, Xin JR, et al. NRN1 and CAT gene polymorphisms, complex noise, and lifestyles interactively affect the risk of noise-induced hearing loss[J]. Biomed Environ Sci, 2021, 34(9): 705-718. doi:10.3967/bes2021.098

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