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Summary
Abstract
Introduction
Protocol
Results
Discussion
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신경과학

通过圆窗膜病毒介导的基因传递给鼠标内耳手术方法

Published: March 16, 2015
doi:
10.3791/52187
, , ,
1Department of Otolaryngology-Head and Neck Surgery,University of California, San Francisco

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

基因疗法，用于实现由感觉神经性耳聋功能恢复，承诺给予更深入的了解，有助于听力损失潜在的分子和遗传机制。导入载体的进入内耳必须以一种方式在整个耳蜗损伤，同时最大限度地降低到现有结构即广泛分布的试剂来完成。这个手稿描述耳后的手术的方法，可以使用分子，药物，和病毒递送至小鼠10日龄和老年人通过圆窗膜（RWM）用于小鼠耳蜗疗法。这种手术方式，可以快速地直接供货到鼓阶，同时尽量减少失血，避免动物的死亡率。此技术涉及到内和中耳的基本的结构以及颈部肌肉，同时完全保留听力忽略的或没有损害。为了证明这一点的手术技术，囊泡glutam的功效吃转运3敲除（VGLUT3 KO）小鼠将被用作先天性耳聋，它回收输送VGLUT3到内耳后使用腺相关病毒（AAV-1）听力小鼠模型的一个例子。

Introduction

基因治疗一直被建议作为对遗传性听力损失的潜在的治疗，但在这方面的成功一直难以实现1。迄今为止，病毒介导的方法学都占优势由于相对难以接近耳蜗内靶向特定细胞类型的理论能力。两个腺病毒（AV）和腺相关病毒（AAV）也已用于耳蜗基因递送。自动增值服务是在耳蜗为若干原因是有利的。它们是复制缺陷型病毒，并且可以有效的转基因的分子转移到不同类型的细胞，包括神经元，为一些听力损失的原因的一个重要目标。 AAV进入细胞是通过特异性受体介导2;因此，特定的血清型的选择必须是兼容的细胞类型，以被转导。自动增值服务可以有效地转染毛细胞3和掺入到宿主基因组中，导致稳定的，长期的TRA的表达nsgenic蛋白和该单元4的表型变化。而对于短期应用，例如毛细胞再生未必是有利的，长期表达是遗传缺陷稳定救援非常重要的。因为自动增值服务不与任何人疾病或感染相关的，并证明无耳毒性5,6,7，它们是在基因治疗中的用途为听力损失8的遗传形式的理想候选。

转移外源遗传物质导入用病毒载体的哺乳动物内耳已经研究在过去十年中，并成为一个有前途的技术，用于治疗遗传和获得性形式的听力损失9。耳蜗是潜在的基因治疗有几个原因的理想目标：1）它的体积小，就必须在有限的所需要的病毒量; 2）从它的其他器官系统的限制副作用相对隔离;及3）其流体填充腔促进病毒交付整个迷宫10，11,12,13,14，15。

先天性耳聋的小鼠模型允许使用的研究方法很多监测系统的，可复制的方式内耳的发展。虽然鼠标耳蜗的小尺寸确实存在一些手术难度，鼠标作为遗传性听力损失的研究具有极其重要的模式，有几个实验优于其他品种16。小鼠模型允许范围内通过遗传连锁分析，收集了详细的形态学观察，并模拟情景致病特点的评估;正因为如此，它们是很好的候选病毒介导的基因治疗。在小鼠中结合的技术进步的广泛的遗传研究已经使得有可能产生转基因小鼠在整个实验室17,18，19，20,21的可重现方式。 FurthermorE，有两个获得性和遗传听力损失表型小鼠，使严格的测试，在这个动物模型22，23,24存在众多的车型。因此，校正听力使用病毒介导的基因治疗在小鼠模型是在寻求一种治疗人类疾病的适当的第一步骤。

之前我们已经表明，缺乏的水泡谷氨酸转运体3（VGLUT3）转基因小鼠天生聋哑，由于缺乏谷氨酸释放的IHC带状突触25。由于此突变不导致感觉毛细胞的初级变性，这些突变小鼠有潜在其中测试耳蜗基因治疗先天性听力损失的优良模型。

迄今为止，一些病毒递送技术耳蜗基因治疗已被描述，包括圆窗膜扩散，圆窗膜注射，并且经由内耳开窗递送。有强大的IAL优点和每种方法9的缺点。

这里，我们报告为通过圆窗膜（RWM）病毒介导的基因递送到VGLUT3 KO小鼠内耳的外科方法。在耳后RWM注射方法是微创具有优良的听证会保留，并且是比较快的。如我们先前发表的，在为了恢复听力在该小鼠模型中，源自于AAV2载体携带VGLUT3基因（AAV1-VGLUT3）引入这些耳聋小鼠的耳蜗在出生后第12天（P！@），导致听26的恢复。听力在VGLUT3 KO小鼠经听性脑干反应（ABR）验证，而转基因蛋白的表达用免疫荧光（IF）的验证。这种方法因而表明病毒介导的基因疗法可以纠正一个遗传缺陷否则将导致耳聋。

Protocol

注：所有的程序和动物处理符合NIH道德准则和加州大学的机构动物护理和使用委员会，旧金山批准的协议要求。 1.准备动物外科手术开展外科手术在干净的专用空间。高压灭菌所有的手术器械，消毒与手术前玻璃珠灭菌。 注：在这个协议中，使用日龄10-12（P10-12）FVB小鼠。不同年龄和小鼠的菌株可用于满足特定项目的需要。年纪比P40小鼠是具有挑战性的，因为泡?…

Representative Results

为了验证该技术的特点和对耳蜗分子治疗耳后的方法实用，AAV1-VGLUT3，AAV1-GFP和AAV2-GFP分别通过RWM送入P10-12小鼠内耳。该方法证明了内毛细胞内成功的转基因表达（IHC）（VGLUT3 图1和GFP 图2和GFP 图3A），外毛细胞（OHC）（GFP 图2）和支持细胞（GFP 图2和图3A）26没有尔蒂伤害显著的器官。表达GFP不同细胞类型的观察看?…

Discussion

在这项工作中，我们详细地描述了可用于耳蜗基因疗法，以恢复或抢救是受遗传缺陷损害正常听觉功能的目标的技术。因为它通常是无创伤，这种方法是安全的耳蜗的基因转移或其他潜在的分子疗法30。其他的方法对于耳蜗疗法已被描述，其中包括一个腹侧方法24，在小鼠和豚鼠内耳开窗31,32和内淋巴囊递送33。在我们的经验中，耳后的方法是更快，更简单，并具有…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

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
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Tags

Gene TherapySensorineural DeafnessInner EarRound Window MembraneCochleaGene DeliveryViral VectorsVGLUT3 Knockout MouseAdeno-associated Virus
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Akil, O., Rouse, S. L., Chan, D. K., Lustig, L. R. Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane. J. Vis. Exp. (97), e52187, doi:10.3791/52187 (2015).
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