Approaches and Vectors for Efficient Cochlear Gene Transfer in Adult Mouse Models

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Quotation shows PackGene:AAV-PHP.eB, AAV-ie, AAV-Anc80L65, AAV2, and AAV-PHP.s were produced by the PackGene Biotech Company (Shanghai, China). All vectors were engineered to express the enhanced green fluorescent protein (eGFP) gene under the control of the chimeric cytomegalovirus (CMV) promoter and carried the woodchuck hepatitis post-transcriptional virus regulatory element (WPRE) cassette. To determine the transduction rate among the different serotypes, we adjusted all the vectors to be at equal doses of 1.0x10^13 VG/mL. Virus aliquots were stored at -80 C and thawed before use.

Research Field:ear

AAV Serotype:AAV-PHP.eB, AAV-ie, AAV-Anc80L65, AAV2, and AAV-PHP.s

Targeted organ:inner ear

Animal or cell line strain:Adult C57BL/6J wild-type mice (4–6 weeks old)

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Abstract

Inner ear gene therapy using adeno-associated viral vectors (AAVs) in neonatal mice can alleviate hearing loss in mouse models of deafness. However, efficient and safe transgene delivery to the adult mouse cochlea is critical for the effectiveness of AAV-mediated therapy. Here, we examined three gene delivery approaches including posterior semicircular canal (PSCC) canalostomy, round window membrane (RWM) injection, and tubing-RWM+PSCC (t-RP) in adult mice. Transduction rates and survival rates of cochlear hair cells were analyzed, hearing function was recorded, AAV distribution in the sagittal brain sections was evaluated, and cochlear histopathologic images were appraised. We found that an injection volume of 1 μL AAV through the PSCC is safe and highly efficient and does not impair hearing function in adult mice, but local injection allows AAV vectors to spread slightly into the brain. We then tested five AAV serotypes (PHP.eB, IE, Anc80L65, AAV2, and PHP.s) in parallel and observed the most robust eGFP expression in inner hair cells, outer hair cells, and spiral ganglion neurons throughout the cochlea after AAV-Anc80L65 injection. Thus, PSCC-injected Anc80L65 provides a foundation for gene therapy in the adult cochlea and will facilitate the development of inner ear gene therapy.

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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.

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