基于腺相关病毒血清型8型介导的Gjb2基因c.109G>A纯合突变耳聋小鼠的基因治疗

doi:10.3969/j.issn.1674-8115.2022.06.007

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (6): 735-741.doi: 10.3969/j.issn.1674-8115.2022.06.007

• 论著 · 基础研究 • 上一篇

基于腺相关病毒血清型8型介导的Gjb2基因c.109G>A纯合突变耳聋小鼠的基因治疗

成桢哲¹^,²^,³(), 金晨曦¹^,²^,³, 冯宝怡¹^,²^,³, 郑晓飞¹^,²^,³, 刘祎晴¹^,²^,³, 吴皓¹^,²^,³(), 陶永¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海市耳鼻疾病转化医学重点实验室，上海 200125
^3.上海交通大学医学院耳科学研究所，上海 200125

收稿日期:2022-02-18 接受日期:2022-06-19 出版日期:2022-06-28 发布日期:2022-08-19
通讯作者: 吴皓,陶永 E-mail:chengzhenzhe@sjtu.edu.cn;wuhao@shsmu.edu.cn;taoyent@foxmail.com
作者简介:成桢哲（1996—），男，硕士生；电子信箱：chengzhenzhe@sjtu.edu.cn。
基金资助:
国家自然科学基金重点项目(81730028)

Adeno-associated virus serotype 8-mediated gene therapy in Gjb2 mutant c.109G>A homozygous mice

CHENG Zhenzhe¹^,²^,³(), JIN Chenxi¹^,²^,³, FENG Baoyi¹^,²^,³, ZHENG Xiaofei¹^,²^,³, LIU Yiqing¹^,²^,³, WU Hao¹^,²^,³(), TAO Yong¹^,²^,³()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200125, China
^3.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China

Received:2022-02-18 Accepted:2022-06-19 Online:2022-06-28 Published:2022-08-19
Contact: WU Hao,TAO Yong E-mail:chengzhenzhe@sjtu.edu.cn;wuhao@shsmu.edu.cn;taoyent@foxmail.com
Supported by:
National Natural Science Foundation of China(81730028)

摘要/Abstract

摘要：

目的·探究腺相关病毒血清型8型（adeno-associated virus serotype 8，AAV8）介导的野生型缝隙连接蛋白β2（gap junction protein β 2，Gjb2）基因在Gjb2基因c.109G>A纯合突变小鼠（简称Gjb2纯合突变小鼠）耳蜗支持细胞区域的表达情况及安全性，以及对呋塞米引起的听力下降的影响。方法·将带有绿色荧光蛋白（green fluorescent protein，GFP）的AAV8-GFP病毒，经耳蜗中阶注射入新生Gjb2纯合突变小鼠的内耳，注射14 d后取小鼠耳蜗，荧光显微镜观察基底膜GFP的表达情况。将载有野生型Gjb2基因的AAV8-GJB2-GFP病毒经中阶注射入新生Gjb2纯合突变小鼠内耳后，于4周后通过实时荧光定量PCR和Western blotting检测耳蜗Gjb2 mRNA及其编码的连接子蛋白（connexin 26，CX26）的表达水平，通过免疫荧光法观察其具体表达位置。通过听性脑干反应（auditory brainstem response，ABR）实验评价新生纯合突变小鼠中阶注射AAV8-GJB2-GFP后4周龄时5.66~45.00 kHz的听力阈值。采用呋塞米试验比较野生型小鼠及Gjb2纯合突变小鼠腹腔注射呋塞米前后的ABR阈值变化，以及观察Gjb2纯合突变小鼠经AAV8-GJB2-GFP治疗后能否缓解呋塞米导致的听力下降。结果·AAV8-GFP注射14 d后耳蜗顶圈、中圈、底圈支持细胞的转染率分别为（11.60±1.28）%、（10.33±1.55）%、（5.40±0.86）%。AAV8-GJB2-GFP注射4周后耳蜗Gjb2 mRNA水平约为未注射耳的1.26倍（P=0.014），CX26蛋白水平是未注射耳的1.31倍（P=0.001）；注射后通过荧光显微镜观察到耳蜗支持细胞表达CX26，内毛细胞区域也有CX26异位表达。ABR检测显示，给药耳各频率听力阈值与对侧耳无明显差异，表明其安全性较好。Gjb2纯合突变小鼠经呋塞米诱导后较野生型小鼠产生更明显的听力阈移，注射AAV8-GJB2-GFP 4周后突变小鼠阈移小于未治疗小鼠，在8.00、11.32、16.00 kHz测得的阈值，2组间差异均有统计学意义（均P<0.05）。结论·在新生Gjb2纯合突变小鼠中阶注射AAV8-GJB2-GFP，可使小鼠成年后耳蜗支持细胞表达外源性Gjb2，挽救呋塞米诱导的听力下降。

Abstract:

Objective·To determine the expression and safety of the wild-type gap junction protein β 2 (Gjb2) gene delivered by adeno-associated virus serotype 8 (AAV8) into cochlear supporting cell region, and its effect on furosemide-induced hearing loss in Gjb2 mutant c.109G>A homozygous mice (Gjb2 homozygous mice).

Methods·The AAV8-GFP viruses with green fluorescent protein (GFP) were injected into the inner ears of newborn Gjb2 homozygous mice through the cochlear scala media, and the GFP expression in the basilar membrane was evaluated by fluorescence microscope 14 d after injection. AAV8-GJB2-GFP viruses with wild-type Gjb2 gene were injected into the inner ears of neonatal Gjb2 homozygous mice, and then the Gjb2 mRNA and its encoded protein connexin 26 (CX26) expression level and location in the cochleae were detected by RT-qPCR, Western blotting and immunofluorescence. The auditory brainstem response (ABR) test was performed to evaluate the hearing thresholds at 5.66?45.00 kHz of the 4-week-old homozygous mice, which were injected with AAV8-GJB2-GFP at the neonatal stage. The furosemide test was used to compare the changes of ABR thresholds before and after intraperitoneal injection in wild-type mice and Gjb2 homozygous mice, and observe whether furosemide-induced hearing loss could be alleviated by AAV8-GJB2-GFP injection in the homozygous mice.

Results·Fourteen days after AAV8-GFP injection, the transfection rates of supporting cells in the apex, middle, and basal turns of the cochleae were (11.60±1.28)%, (10.33±1.55)%, and (5.40±0.86)%. Four weeks after AAV8-GJB2-GFP injection, the Gjb2 mRNA expression level was 26% higher than that in the uninjected side, and CX26 protein level was 31% higher than that in the uninjected side. The expression of CX26 protein was observed in the supporting cells and ectopic expression of CX26 was also observed between the inner hair cells by using fluorescent microscope. The ABR thresholds did not shift after AAV8-GJB2-GFP injection, indicating its safety. Gjb2 homozygous mice exhibited worse hearing loss than wild-type mice after furosemide injection, whereas AAV8-GJB2-GFP alleviated furosemide-induced hearing loss in Gjb2 homozygous mice, and the thresholds measured at 8.00, 11.32 and 16.00 kHz were significantly different between the treated mice and the untreated ones (P<0.05).

Conclusion·The injection of AAV8-GJB2-GFP into the scala media of neonatal Gjb2 homozygous mice can make the cochlear supporting cells express exogenous Gjb2 in the adulthood, which alleviate the furosemide-induced hearing loss.

Key words: adeno-associated virus (AAV), gap junction protein beta 2 (Gjb2), supporting cell, furosemide, gene therapy

中图分类号:

R764.43

成桢哲, 金晨曦, 冯宝怡, 郑晓飞, 刘祎晴, 吴皓, 陶永. 基于腺相关病毒血清型8型介导的Gjb2基因c.109G>A纯合突变耳聋小鼠的基因治疗[J]. 上海交通大学学报（医学版）, 2022, 42(6): 735-741.

CHENG Zhenzhe, JIN Chenxi, FENG Baoyi, ZHENG Xiaofei, LIU Yiqing, WU Hao, TAO Yong. Adeno-associated virus serotype 8-mediated gene therapy in Gjb2 mutant c.109G>A homozygous mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(6): 735-741.

图/表 6

表1 实时荧光定量PCR引物序列

Tab 1 Primer sequences for real-time qPCR

Name	Sequence（5’→3’）
Gjb2-Forward	ATCCTCGGGGGTGTCAACAA
Gjb2-Reverse	AGACAAAATCGGCTTGCTCATC
Gjb6-Forward	ACCAGCATAGGGAAGGTGTG
Gjb6-Reverse	TGCAGAGTGTTGCAGACAAAG
Rpl19-Forward	ACCTGGATGAGAAGGATGAG
Rpl19-Reverse	ACCTTCAGGTACAGGCTGTG

图1 Gjb2 纯合突变小鼠耳蜗中阶注射AAV8-GFP 14 d后支持细胞转染情况Note：A. Representative images of GFP transfection in supporting cells of homozygous mice (middle turn). B. Statistical results of virus transfection in the apex, middle and basal turns of the cochleae in homozygous mice.

Fig 1 Supporting cells transfection 14 d after AAV8-GFP injection in the scala media of the cochleae of Gjb2 homozygous mice

图2 Gjb2 纯合突变小鼠耳蜗中阶注射AAV8-GJB2-GFP 4周后 Gjb2 及其蛋白CX26在耳蜗的表达和定位Note：A. mRNA expression levels of Gjb2 and Gjb6 detected by RT-qPCR(n=3); B. Western blotting results and statistics of CX26 expression (n=3). C. CX26 (with GFP) expression in the basilar membrane of the top turn of the cochlea. D. Ectopic expression of CX26 (with GFP) between the hair cells observed under high magnification. ①P=0.014, ②P=0.001.

Fig 2 Expression and location of Gjb2 and it's expression protein CX26 in the cochleae of Gjb2 homozygous mice 4 weeks after AAV8-GJB2-GFP injection in the scala media

图3 Gjb2 纯合突变小鼠一侧耳蜗中阶注射AAV8-GJB2-GFP 4周后ABR阈值与未注射侧的比较

Fig 3 Comparison of ABR thresholds between AAV8-GJB2-GFP injected side and contralateral side of cochleae after 4 weeks in Gjb2 homozygous mice

图4 4周龄野生型小鼠及 Gjb2 纯合突变小鼠注射呋塞米前后ABR阈值变化Note：WT—wild type mice; Homo—Gjb2 homozygous mice.①P=0.000, vs Homo.

Fig 4 Changes of ABR thresholds before and after furosemide injection in 4-week-old wild type mice and Gjb2 homozygous mice

图5 Gjb2 纯合突变小鼠注射AAV8-GJB2-GFP 4周后在呋塞米试验中的ABR阈值变化Note：①P=0.037, ②P=0.028, ③P=0.009.

Fig 5 Changes of ABR thresholds in furosemide test in the Gjb2 homozygous mice 4 weeks after AAV8-GJB2-GFP injection

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基于腺相关病毒血清型8型介导的Gjb2基因c.109G>A纯合突变耳聋小鼠的基因治疗

Adeno-associated virus serotype 8-mediated gene therapy in Gjb2 mutant c.109G>A homozygous mice

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