Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

Li Xia,Shankai Yin,Jian Wang,Andreas R Janecke

doi:10.1371/journal.pone.0043218

Li Xia, Shankai Yin + Show 2 more

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https://doi.org/10.1371/journal.pone.0043218

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Abstract

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant.

Highlights

Gene therapy is the process of insertion, alteration, and/or removal of targeted genes to and from an individual’s cells to treat medical conditions
We reported the use of a newly developed adenoassociated virus with mutations of surface-exposed tyrosine residues [15], in combination with digestion of the round window membrane (RWM) to achieve inner ear gene transfection in guinea pigs
The investigation of gene therapy in mice is justified by the importance of mouse models in the research of genetic hearing loss and the early onset of this loss in many cases

Summary

Introduction

Gene therapy is the process of insertion, alteration, and/or removal of targeted genes to and from an individual’s cells to treat medical conditions. Genetic manipulation at the genome level (germ line gene therapy) is not considered ethical because of unpredictable effects, and genetic manipulation in somatic cells must be restrictively controlled to reduce the associated risks [1]. Local gene transfection to a limited region is one of the most convenient ways to enable such control. The cochlea is traditionally considered as an ideal organ for local gene therapy because of its anatomical isolation and the fluid streaming internally [2]. The isolation may limit the risk of side effects that may occur in other tissues and organs (e.g., via the neoplastic process), and fluid movement may facilitate transportation of the targeted gene [3,4,5]. The effectiveness of this isolation has not been comprehensively evaluated in the situation of virus mediated cochlear gene transfection

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