This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

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
<ol>
	<li>Carry out surgical procedures in a clean, dedicated space. Autoclave all surgical instruments, sterilize with a glass-bead sterilizer&#160;prior to surgery. 
	NOTE: In this protocol, use postnatal day 10-12 (P10-12) FVB mice. Differen...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlea+microinjection+and+its+effects+upon+auditory+function+in+guinea+pig.">Cochlea microinjection and its effects upon auditory function in guinea pig.</a> Eur Arch Otorhinolaryngol. 257 (9), 469-472 (2000).</li><li>Iizuka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+in+vivo+delivery+of+transgene+via+adeno-associated+virus+into+supporting+cells+of+the+neonatal+mouse+cochlea.">Noninvasive in vivo delivery of transgene via adeno-associated virus into supporting cells of the neonatal mouse cochlea.</a> Hum. Gene Ther. 19 (4), 384-390 (2008).</li><li>Kilpatrick, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus-mediated+gene+delivery+into+the+scala+media+of+the+normal+and+deafened+adult+mouse+ear.">Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.</a> Gene Ther. 18 (6), 569-578 (2011).</li><li>Yamasoba, T., Yagi, M., Roessler, B. J., Miller, J. M., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inner+Ear+Transgene+Expressionafter+Adenoviral+Vector+Inoculation+in+the+Endolymphatic+Sac+Hum.">Inner Ear Transgene Expressionafter Adenoviral Vector Inoculation in the Endolymphatic Sac Hum.</a> Gene Ther. 10 (5), 769-774 (1999).</li><li>Praetorius, M., Baker, K., Weich, C. M., Plinkert, P. 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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, cochleostomy31,32 and endolymphatic sac delivery33 in mouse and guinea p...

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) &#160;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 the cell is mediated by specific receptors2; thus, the choice of a particular serotype must be compatible with the cell types to be transduced. AAVs can effectively transfect hair cells3 and incorporate into the host genome, resulting in stable, long-term expression of the transgenic protein and phenotypic change in the cell4. While not necessarily advantageous for short-term applications such as hair-cell regeneration, long-term expression is very important for stable rescue of genetic defects. Because AAVs are not associated with any human disease or infection and demonstrate no ototoxicity5,6,7, they are an ideal candidate for use in gene therapy for inherited forms of hearing loss8.
Transfer of exogenous genetic material into the mammalian inner ear using viral vectors has been studied over the last decade and is emerging as a promising technique for treating both genetic and acquired forms of hearing loss9. The cochlea is potentially an ideal target for gene therapy for several reasons: 1) its small volume necessitates&#160;a limited amount of the virus needed; 2) its relative isolation from other organ systems limits side effects; and 3) its fluid-filled chambers facilitate viral delivery throughout the labyrinth10,11,12,13,14,15.
Mouse models of congenital deafness allow for use of many methods of study to monitor development of the inner ear in a systematic, replicable way. While the small size of mouse cochleae does present some surgical difficulty, the mouse serves as an extremely important model in the study of genetic hearing loss, with several experimental advantages over other species16. Mouse models allow assessment of a range of characteristics through genetic linkage analysis, collection of detailed morphological observations, and simulating pathogenic scenarios; as such, they are good candidates for virally mediated gene therapy. Extensive genetic studies in mice combined with technological advances have made it possible to generate genetically modified mice in a reproducible way across laboratories17,18,19,20,21. Furthermore, there exist numerous models for both acquired and inherited hearing loss phenotypes in mice, allowing rigorous testing in this animal model22,23,24. Thus, correcting hearing using virally mediated gene therapy in a mouse model is an appropriate first step in the search for a cure for human disease.
We have previously shown that transgenic mice lacking vesicular glutamate transporter 3 (VGLUT3) are born deaf due to lack of glutamate release at the IHC ribbon synapse25. Because this mutation does not lead to a primary degeneration of the sensory hair cells, these mutant mice are &#160;potentially an excellent model in which to test cochlear gene therapy for congenital hearing loss.
To date, a number of viral delivery techniques for cochlear gene therapy have been described, including round window membrane diffusion, round window membrane injection, and delivery via a cochleostomy. There are potential advantages and disadvantages of each of these approaches9.
Here we report a surgical method for virally mediated gene delivery to the VGLUT3 KO mouse inner ear through the round window membrane (RWM). The post-auricular RWM injection method is minimally invasive with excellent hearing preservation, and is relatively fast. As we have previously published, in an effort to restore hearing in this mouse model, an AAV1 vector carrying the VGLUT3 gene (AAV1-VGLUT3) was introduced into the cochlea of these deaf mice at postnatal day 12(P!@), resulting in the restoration of hearing26. Hearing in the VGLUT3 KO mice was verified by auditory brainstem response (ABR), while transgene protein expression was verified using immunofluorescence (IF). This methodology thus demonstrates that virally-mediated gene therapy can correct a genetic defect that would otherwise results in deafness.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Name</td>
			<td style="width:177px;">Company</td>
			<td style="width:161px;">Catalog Number</td>
			<td style="width:111px;">Comments</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Ketamine</td>
			<td style="width:177px;">Butler Schein</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Xylazine</td>
			<td style="width:177px;">AnaSed</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Acepromazine</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Carprofen analgesia</td>
			<td style="width:177px;">Provided by UCSF LARC</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:355px;">Betadine</td>
			<td style="width:177px;">Betadine Puredue Pharma</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">dexamethasone ophthalmic ointment (TobraDex)</td>
			<td style="width:177px;">Alcon</td>
			<td style="width:161px;"></td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:355px;">Heating pad</td>
			<td style="width:177px;">Braintree scientific, inc.</td>
			<td style="width:161px;"></td>
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			<td style="width:177px;">BD</td>
			<td align="right" style="width:161px;">305127</td>
			<td style="width:111px;"></td>
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			<td height="40" style="height:40px;width:355px;">Borosilicate capillary pipette</td>
			<td style="width:177px;">World precision instruments, inc.</td>
			<td style="width:161px;">1B100F-4</td>
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			<td style="width:177px;">Butler Schein</td>
			<td align="right" style="width:161px;">31477</td>
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			<td>Leica</td>
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			<td height="20" style="height:20px;">Flaming/ Brown Micropipette&#160;&#160;&#160;&#160;&#160;</td>
			<td>Sutter Instrument Co</td>
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			<td height="20" style="height:20px;">Puller Model P-97&#160;&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;">TDT BioSig III System&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>&#160;Tucker-Davis Technologies</td>
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	</tbody>
</table>

surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

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).

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</stro...

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

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.

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.0K 조회수.  University of California, San Francisco. 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. 