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Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues.

ABSTRACT: Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.

SUBMITTER: Casey G

PROVIDER: S-EPMC7682903 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues.

Casey Graham G Askew Charles C Brimble Mark A MA Samulski R Jude RJ Davidoff Andrew M AM Li Chengwen C Walters Bradley J BJ

PloS one 20201123 11

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage ...[more]

PMID: 33227033

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Project description:Adeno-associated virus (AAV) is the preferred vector for gene therapy of hereditary deafness, and different viral serotypes, promoters and transduction pathways can influence the targeting of AAV to different types of cells and the expression levels of numerous exogenous genes. To determine the transduction and expression patterns of AAV with different serotypes or promoters in hair cells and supporting cells in the neonatal mouse cochlea, we examined the expression of enhanced green fluorescent protein (eGFP) for five different types of AAV vectors [serotypes 2, 9, and Anc80L65 with promoter cytomegalovirus (CMV)-beta-Globin and serotypes 2 and 9 with promoter chicken beta-actin (CBA)] in in vitro cochlear explant cultures and we tested the transduction of AAV2/2-CBA, AAV2/9-CBA, and AAV2/Anc80L65-CMV by in vivo microinjection into the scala media of the cochlea. We found that each AAV vector had its own transduction and expression characteristics in hair cells and supporting cells in different regions of the cochlea. There was a tonotopic gradient for the in vitro transduction of AAV2/2-CBA, AAV2/9-CBA, AAV2/2-CMV, and AAV2/9-CMV in outer hair cells (OHCs), with more OHCs expressing eGFP at the base of the cochlea than at the apex. AAV2/2-CBA in vitro and AAV2/Anc80L65-CMV in vivo induced more supporting cells expressing eGFP at the apex than in the base. We found that AAV vectors with different promoters had different expression efficacies in hair cells and supporting cells of the auditory epithelium. The CMV-beta-Globin promoter could drive the expression of the delivered construct more efficiently in hair cells, while the CBA promoter was more efficient in supporting cells. The in vitro and in vivo experiments both demonstrated that AAV2/Anc80L65-CMV was a very promising vector for gene therapy of deafness because of its high transduction rates in hair cells. These results might be useful for selecting the appropriate vectors for gene delivery into different types of inner ear cells and thus improving the effectiveness of gene therapy.

Dataset Information

Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues.

Publications

Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues.

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