Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Summary

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Abstract

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

Introduction

Gene therapy has long been suggested as a potential treatment for genetic hearing loss, but success in this area has remained elusive¹. To date, virally mediated methodologies have predominated due the theoretic ability to target specific cell types within the relatively inaccessible cochlea. Both adenovirus (AV) and adeno-associated virus (AAV)  have been used for cochlear gene delivery. AAVs are advantageous in the cochlea for a number of reasons. They are replication-deficient viruses and can efficiently transfer transgenic molecules to different cell types including neurons, an important target for a number of causes of hearing loss. AAV entry into....

Protocol

NOTE: All procedures and animal handling complied with NIH ethics guidelines and approved protocol requirements of the Institutional Animal Care and Use Committee of the University of California, San Francisco.

1. Preparing the Animal for Surgery

1. Carry out surgical procedures in a clean, dedicated space. Autoclave all surgical instruments, sterilize with a glass-bead sterilizer prior to surgery.
   NOTE: In this protocol, use postnatal day 10-12 (P10-12) FVB mice. Different ages and strains of mice can be used to meet the needs of a specific project. Mice older than P40 can be challenging because the bulla bone gets har....

Results

To verify the technical features and utility of the post-auricular approach for cochlear molecular therapy, AAV1-VGLUT3, AAV1-GFP and AAV2-GFP were delivered into P10-12 mice inner ear via the RWM. This approach demonstrates successful transgene expression within inner hair cells (IHC) (VGLUT3 Figure 1 and GFP Figure 2 and GFP Figure 3A), outer hair cells (OHC) (GFP Figure 2) and supporting cells (GFP Figure 2 and Figure 3A

Discussion

In this work, we describe in detail a technique that can be used for cochlear gene therapy, with the goal of restoring or rescuing normal auditory function that is compromised by a genetic defect. As it is typically atraumatic, this approach is safe for cochlear gene transfer or other potential molecular therapies³⁰. Other approaches for cochlear therapy have been described, including a ventral approach²⁴, cochleostomy^31,32 and endolymphatic sac delivery³³ in mouse and guinea p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine	Butler Schein
Xylazine	AnaSed
Acepromazine	Provided by UCSF LARC
Carprofen analgesia	Provided by UCSF LARC
Betadine	Betadine Puredue Pharma
dexamethasone ophthalmic ointment (TobraDex)	Alcon
Heating pad	Braintree scientific, inc.
25G needle	BD	305127
Borosilicate capillary pipette	World precision instruments, inc.	1B100F-4
Suture PDS*plus Antibacterial	Ethicon	PDP149
Tissue glue (Vetcode)	Butler Schein	31477
Rabbit Anti-GFP antibody	Invitrogen	A11122
Dissecting microscope	Leica		MZ95
Flaming/ Brown Micropipette	Sutter Instrument Co
Puller Model P-97
TDT BioSig III System	Tucker-Davis Technologies