Front Mol Neurosci. 2022 Oct 19
Zhong-Rui Chen, Jing-Ying Guo, Lu He, Shan Liu, Jun-Yi Xu, Zi-Jing Yang, Wei Su, Ke Liu, Shu-Sheng Gong, and Guo-Peng Wang
Products used in the paper Details Operation
AAV-ie with reporter genes
of enhanced green fluorescent protein (EGFP) or mCherry
We used purified AAV-ie viral vectors driven by the promoters of cytomegalovirus (CMV) or CMV early enhancer/chicken b-actin (CAG). AAV-ie with reporter genes of enhanced green fluorescent protein (EGFP) or mCherry were used for the experiments. The vectors were purchased from PackGene Biotech, Inc. at a titer of 1*10^13 vg/mL. The vectors were generated by triple plasmid transfection into HEK293T cells. The titers of the vectors were determined using Droplet Digital PCR. Vector aliquots were stored in phosphate-buffered saline (PBS) with 0.001% pluronic F-68 at -80℃. Request Quote

Research Field: CNS

Keywords: adeno-associated virus, gene transfer, utricle, mice, hair cell, transduction

AAV Serotype: AAV-ie

Dose: Mixed vector suspensions were inoculated via the posterior semicircular canal at a rate of 0.5 ml/min using a micro-injection pump. For the sequential administration of dual vectors, 1 ml of AAV-ie-CAG-EGFP vector was injected through the lateral semicircular canal, followed 1 week later by a second injection of 1 ml of AAVie-CAG-mCherry through the posterior semicircular canal.

Routes of Administration: Dual AAV-ie-CAG vectors (AAV-ie-CAG-EGFP and AAV-ie-CAG-mCherry) or dual AAV-ie-CMV vectors (AAV-ie-CMV-EGFP and AAV-ie-CMV-mCherry) were inoculated into the inner ear of adult mice. Utricles were harvested 2 weeks after the surgery.

Animal or cell line strain: Wild-type C57BL/6J (6–8-weekold) and CD-1 (postnatal day 1, P1) mice were purchased from
SPF Biotechnology Co., Ltd. (Beijing, China). Pou4f3C=DTR mice were purchased from the Jackson Laboratory

Abstract

Adeno-associated virus (AAV)-mediated gene transfer is an efficient method of gene over-expression in the vestibular end organs. However, AAV has limited usefulness for delivering a large gene, or multiple genes, due to its small packaging capacity (< 5 kb). Co-transduction of dual-AAV vectors can be used to increase the packaging capacity for gene delivery to various organs and tissues. However, its usefulness has not been well validated in the vestibular sensory epithelium. In the present study, we characterized the co-transduction of dual-AAV vectors in mouse utricles following inoculation of two AAV-serotype inner ear (AAV-ie) vectors via canalostomy. Firstly, co-transduction efficiencies were compared between dual-AAV-ie vectors using two different promoters: cytomegalovirus (CMV) and CMV early enhancer/chicken β-actin (CAG). In the group of dual AAV-ie-CAG vectors, the co-transduction rates for striolar hair cells (HCs), extrastriolar HCs, striolar supporting cells (SCs), and extrastriolar SCs were 23.14 ± 2.25%, 27.05 ± 2.10%, 57.65 ± 7.21%, and 60.33 ± 5.69%, respectively. The co-transduction rates in the group of dual AAV-ie-CMV vectors were comparable to those in the dual AAV-ie-CAG group. Next, we examined the co-transduction of dual-AAV-ie-CAG vectors in the utricles of neonatal mice and damaged adult mice. In the neonatal mice, co-transduction rates were 52.88 ± 3.11% and 44.93 ± 2.06% in the striolar and extrastriolar HCs, respectively, which were significantly higher than those in adult mice. In the Pou4f3+/DTR mice, following diphtheria toxin administration, which eliminated most HCs and spared the SCs, the co-transduction rate of SCs was not significantly different to that of normal utricles. Transgene expression persisted for up to 3 months in the adult mice. Furthermore, sequential administration of two AAV-ie-CAG vectors at an interval of 1 week resulted in a higher co-transduction rate in HCs than concurrent delivery. The auditory brainstem responses and swim tests did not reveal any disruption of auditory or vestibular function after co-transduction with dual-AAV-ie vectors. In conclusion, dual-AAV-ie vectors allow efficient co-transduction in the vestibular sensory epithelium and facilitate the delivery of large or multiple genes for vestibular gene therapy.

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