Co-transduction of dual-adeno-associated virus vectors in the neonatal and adult mouse utricles
Brief intro:
- Author: 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
- Journal: Front Mol Neurosci
- Doi: https://www.doi.org/10.3389/fnmol.2022.1020803
- Publication Date: 2022 Oct 19
Products/Services used in the paper
Quotation shows PackGene: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 Co., Ltd. (Guangzhou, Guangdong, China) 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℃.
Research Field:CNS
AAV Serotype:AAV-ie
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.
About PackGene
PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.
