Comparison of inner hair cell transduction by different serotypes of adeno-associated virus via cisterna magna injection in mice

doi:10.3969/j.issn.1674-8115.2026.07.008

Abstract

Abstract:

Objective ·To evaluate the safety of cisterna magna-delivered adeno-associated virus (AAV) of different serotypes carrying the human cytomegalovirus immediate-early promoter (CMV) in the auditory and central nervous systems, as well as their transduction differences in inner hair cells (IHCs) and the central nervous system. Methods ·Eight AAV serotypes (AAV1, AAV2, AAV8, AAV9, AAV-rh10, AAV-DJ, AAV-PHP.B, and AAV-PHP.eB) carrying enhanced green fluorescent protein (EGFP) were injected into the cerebrospinal fluid of 4-week-old wild-type mice via cisterna magna. Auditory brainstem response (ABR) was measured to evaluate changes in auditory thresholds before injection and 2 weeks after injection. At 2 weeks post-injection, cochleae were collected and subjected to immunofluorescence staining to compare the transduction efficiency and number of cochlear IHCs among the eight AAV-EGFP serotypes. The EGFP fluorescence signal intensity of the three serotypes with higher transduction efficiency was further analyzed in IHCs. Fluorescent DNA dye staining on frozen brain sections was used to compare the central nervous system transduction characteristics of the three serotypes. Open-field test was performed to assess the effects of cisterna magna injection of three AAV-EGFP serotypes on central nervous system safety in mice. Results ·ABR tests showed no significant differences in auditory thresholds at all tested frequencies before and 2 weeks after cisterna magna injection. Immunofluorescence staining demonstrated that all eight AAV-EGFP serotypes successfully transduced IHCs in the 32 kHz cochlear region at 2 weeks after in jection. Among them, AAV2-CMV-EGFP [(83.33±14.79)%], AAV-DJ-CMV-EGFP [(82.52±5.18)%], and AAV-PHP.B-CMV-EGFP [(96.61±1.87)%] exhibited relatively high transduction efficiency in IHCs at 32 kHz, and no significant intergroup difference was observed in the number of IHCs after injection. Analysis of EGFP fluorescence intensity in the three serotypes with the highest transduction efficiency revealed stronger fluorescence signals in IHCs transduced by AAV-PHP.B-CMV-EGFP and AAV2-CMV-EGFP at the 32 kHz region. Staining of frozen brain sections revealed distinct transduction patterns of the three AAV-EGFP serotypes in the central nervous system following cisterna magna injection. AAV2-CMV-EGFP exhibited relatively low transduction levels; AAV-DJ-CMV-EGFP showed detectable transduction in the cerebellar cortex, dorsal medulla, and periventricular regions; AAV-PHP.B-CMV-EGFP showed transduction in the molecular and granular layers of the cerebellar cortex and periventricular regions. Open-field test results indicated that spontaneous locomotor activity of mice remained normal following cisterna magna injection of the three tested viral serotypes. Conclusion ·Cisterna magna injection of different AAV-EGFP serotypes causes neither hearing impairment nor IHCs injury in mice. Different AAV-EGFP serotypes exhibit distinct transduction efficiencies in the cochlea and central nervous system.

Key words: gene therapy, adeno-associated virus (AAV), cisterna magna injection, inner hair cell (IHC)

CLC Number:

R764.3

An Yiran, Feng Baoyi, Sun Yilin, Wu Hao, Tao Yong. Comparison of inner hair cell transduction by different serotypes of adeno-associated virus via cisterna magna injection in mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(7): 896-905.

Figures/Tables 4

Fig 1 Schematic illustration of cisterna magna injection and auditory safety evaluation in mice 2 weeks after AAV-EGFP administration

Fig 2 Analysis of transduction efficiency and number of IHCs in the 32 kHz cochlear region of mice at 2 weeks after cisterna magna injection of 8 AAV-EGFP serotypes

Fig 3 Comparison of fluorescence intensity and transduction efficiency of AAV2-CMV-EGFP, AAV-DJ-CMV-EGFP, and AAV-PHP.B-CMV-EGFP in mouse cochlear IHCs after cisterna magna injection

Fig 4 Transduction patterns in central nervous system regions and safety effects of AAV2-CMV-EGFP, AAV-DJ-CMV-EGFP, and AAV-PHP.B-CMV-EGFP following cisterna magna injection

TrendMD

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