Name: subconjunctival administration of adeno-associated virus vectors in small animal models
Uploaded: 2022-03-16T14:15:00
Description: Watch this Scientific Journal Video about Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models at JoVE.com

作者感谢北卡罗来纳大学的Vector Core提供本研究中使用的scAAV8-GFP载体，CGIBD组织学核心以及Brian C. Gilger博士的实验室为本研究的临床评估方面提供帮助。这项研究得到了辉瑞-北卡罗来纳州生物技术杰出博士后奖学金和美国基因与细胞治疗学会和囊性纤维化基金会的职业发展奖的支持。内容完全由作者负责，并不一定代表美国基因与细胞治疗学会或囊性纤维化基金会的官方观点。

所有动物程序均按照北卡罗来纳大学教堂山分校机构动物护理和使用委员会的规定进行。使用AAV载体是生物安全1级生物危害风险。处理AAV时，请穿戴适当的个人防护设备，包括实验室外套，手套和护目镜。对于本文描述的实验，利用与血清型8衣壳包装的重组AAV载体，并编码控制绿色荧光蛋白（GFP）表达的通用巨细胞病毒（CMV）启动子。
1. AAV病媒处理和储存
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<ol>
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AAV介导的基因疗法在治疗眼部疾病方面具有巨大潜力。目前的眼部基因治疗依赖于两种主要的局部给药途径，玻璃体内和视网膜下注射。不幸的是，这两种途径都是侵入性的，可能导致严重的并发症，包括视网膜脱离、白内障形成和眼内炎。因此，研究相对较少的侵入性途径，例如SCJ注射，是非常有趣的。
虽然这种技术相对简单，侵入性明显较小，但必须强调AAV递送的几个?...

眼部疾病包括广泛的遗传和获得性疾病。2015年，全世界估计有3600万人在法律上失明，超过10亿人至少患有一定程度的视力障碍，这突出表明有必要在各级扩大缓解工作1。提供眼部药物的主要方法包括局部和局部给药，例如滴眼液或结膜下 （SCJ）、前房内、玻璃体内和视网膜下注射。尽管无创局部治疗是眼科药物最常见的给药方法，并且广泛用于许多眼前节疾病，但角膜解剖屏障的存在对局部给药物质的生物利用度、生物分布和疗效提出了挑战，这表明它可能不是许多内眼疾病的最佳候选治疗途径。局部注射到受疾病影响的特定眼室可能是一种更有效和有针对性的药物递送方法2。然而，重复注射引起的不良反应会使给药策略复杂化。理想情况下，治疗应在单次给药后保持长期疗效。因此，基因治疗是一种有前途的选择，可以最大限度地减少所需注射次数并为治疗眼部疾病提供持续的转基因表达3，4。
许多病毒和非病毒载体可用于基因治疗;然而，AAV载体因其出色的安全性而受到高度关注。AAV 是一种小的、单链的、无包膜的 DNA 病毒，最初于 1965 年由 Atchison 等人发现为腺病毒制剂的污染物5，6 AAV 随后在 1980 年代被设计为基因传递的有效病毒载体，并已成为许多疾病（包括眼部疾病）的首选基因治疗载体， 在过去的几十年里。其中最引人注目的是第一个市售基因治疗药物voretigene neparvovec，它被美国食品和药物管理局批准用于治疗Leber的先天性黑朦，这是一种罕见的后眼病。尽管沃雷蒂基因已成功地克服了临床开发的障碍，但其他眼部基因疗法的商业化仍面临挑战。例如，将voretigene neparvovec施用于通过视网膜下注射保留活视网膜细胞的患者。因此，缺乏活视网膜细胞的更晚期疾病患者没有资格接受治疗，因为它不会提供临床益处。此外，还观察到与视网膜下注射程序相关的已知并发症，包括眼部炎症，白内障，视网膜撕裂，黄斑病变和疼痛7，8。与此手术相关的其他问题包括出血，视网膜脱离，眼内炎的可能性，以及通过眼组织破坏撤销眼部免疫特权状态的可能性9，10，11，12。因此，探索侵入性较小的基因递送途径（例如SCJ注射）的努力变得越来越重要13，14，15，16，17。
结膜是包含3-5层细胞的薄膜，连接前眼和内眼睑。SCJ注射剂在临床上用于将眼科药物输送到眼睛的前段和/或后段，以治疗眼部疾病，例如年龄相关性黄斑变性，青光眼，视网膜炎和后葡萄膜炎18，19。它们相对容易执行，常规用于门诊环境中的眼科药物输送20，有点无痛，不会损害眼部免疫特权，并允许给药药物通过包含视神经的大眶周区域扩散。因此，SCJ注射是AAV基因治疗应用的一种有吸引力的给药途径。以前已经对通过SCJ注射在小鼠中施用的天然AAV血清型的安全性，转导效率，血清免疫原性，生物分布和组织特异性进行了表征13，16，21。这些数据表明，通过SCJ给药将基因传递到单个眼组织是一种正式的可能性。
本文描述了一种简单且适应性强的SCJ注射方案，以在小鼠模型中递送AAV载体。为了确保该方法的可重复性，描述了由体视显微镜、可编程输液/取液注射泵（允许一致和精确的注射速度和压力）和气密可拆卸注射器与显微注射针头耦合的注射系统。该系统适用于其他眼内给药途径，例如小动物的基质内、前房内、玻璃体内和视网膜下注射。此外，荧光素染料通常用于AAV注射部位的可视化。将详细讨论给药路线中的关键实际步骤、注射平台的设置、注射的制备以及直接经验的提示。最后，将简要讨论用于确认AAV递送到所需组织的常见验证技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>36 G NanoFil Needles</td>
			<td>World Precision Instruments</td>
			<td>NF36BV-2</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV vector</td>
			<td>&#160; University of North Carolina at Chapel Hill</td>
			<td>&#160; /</td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Henry Schein</td>
			<td>NDC 11695-0079-8</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP antibody</td>
			<td>AVES labs Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Cannon</td>
			<td>Cannon EOS T5i</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA/RNA extraction kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Forceps</td>
			<td>Fine Science Tools</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7654-01</td>
			<td></td>
		</tr>
		<tr>
			<td>India ink</td>
			<td>StatLab</td>
			<td>NC9903975</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride injection solution</td>
			<td>Henry Schein</td>
			<td>NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Moisture-resistant film</td>
			<td>Parafilm</td>
			<td>807-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Becton Dickinson and Company</td>
			<td>427401</td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine 0.1%</td>
			<td>Bausch Health US</td>
			<td>NDC 24208-730-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Rebound tonometer</td>
			<td>Tonovet</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluorescein solution</td>
			<td>Sigma-Aldich</td>
			<td>46960</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Infuse/Withdraw Pump 11 Pico Plus Elite Programmable Syringe Pump</td>
			<td>Harvard Bioscience</td>
			<td>70-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscopye</td>
			<td>Leica</td>
			<td>Mz6</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracaine Hydrochloride Ophthalmic Solution 0.5%</td>
			<td>Bausch and Lomb</td>
			<td>Rx only</td>
			<td></td>
		</tr>
		<tr>
			<td>Topical ointment</td>
			<td>GenTeal</td>
			<td>NDC 0078-0429-47</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Zone-Quick Phenol Red Thread Box 100 Threads</td>
			<td>ZONE-QUICK</td>
			<td>PO6448</td>
			<td></td>
		</tr>
	</tbody>
</table>

subconjunctival administration of adeno-associated virus vectors in small animal models

眼部疾病包括广泛的遗传性和获得性疾病，由于它们相对容易通过多种给药途径获得，因此是局部药物递送的吸引力靶标。结膜下（SCJ）注射比其他眼内给药途径具有优势，因为它们简单，安全，并且通常在门诊进行。由于眼睛的大小，小动物的SCJ注射通常需要手术显微镜的协助。先前的工作表明，SCJ注射特异性腺相关病毒（AAV）血清型是一种有效的基因传递策略，用于靶向转导眼表，眼肌，角膜和视神经，为治疗许多眼部疾病提供了潜在的方法。
在此，提出了使用由可编程输液/戒断注射泵（允许一致和精确的注射速度和压力）和气密可拆卸注射器与显微注射针头耦合的注射系统组成的小鼠模型中SCJ注射的详细方案。该注射系统还适用于其他眼内给药途径，例如小动物的基质内、前房内、玻璃体内和视网膜下注射。尽管描述了用于眼部基因治疗研究的腺相关病毒载体的递送，但本文中的方案也可以适用于小动物模型中的各种眼科溶液。将详细讨论给药路线中的关键实际步骤、注射平台的设置、注射的制备以及直接经验的提示。此外，还将简要讨论用于AAV递送确认到所需组织的常见验证技术。

注射到结膜下间隙的溶液根据注射量表现为水疱。 在该实验中，在立体显微镜下用36 G针头注射7 μL AAV（7 ×10 9 病毒基因组（vg）/眼）与荧光素混合，终浓度为0.1%，并使用可编程注射泵保持注射速度/压力恒定1μL / s。注射时可能会出现水泡（箭头）。将AAV载体施用到小鼠SCJ室的显微镜视图如图 1所示。
注射到结膜下间隙的物...

Watch this Scientific Journal Video about Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models at JoVE.com

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

在本手稿中，结膜下注射被证明是一种有效的小鼠眼组织载体递送方法，使用由输液/戒断注射泵和气密可拆卸注射器与显微注射针头组成的注射系统。该注射系统也适用于其他眼内给药途径。

小动物模型中腺相关病毒载体的结膜下给药

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

该协议提供了一种以安全，简单和有效的方式将基因疗法递送到多个眼组织的方法。它也适用于小动物的基质内和视网膜下注射。使用结膜下注射途径是微创的。它带来的风险较小，并且对于AV输送治疗多种眼部疾病具有很大的希望。全世界有超过10亿人患有视力障碍，使用安全简便的药物输送方法为治疗许多不同原因的失明提供了广泛的适用性。该协议中描述的基因递送方法对于那些正在开发潜在干预策略和执业眼科医生的人来说特别感兴趣，他们最终将这些药物交付给患者。对于第一次执行此协议的人来说，最大的挑战可能是将针插入概念结膜空间。首先，通过将体视显微镜和注射泵放置在生物安全柜中来组装注射系统。接下来，将聚乙烯管切割成大约 50 厘米的长度。将 36 号针头的末端插入管子的一端。然后用无菌水填充一次性3毫升注射器，并将其插入针头对面的管子侧面。冲洗管道三次，在无菌水和70%酒精之间交替对管道进行消毒，并确保整个管道没有泄漏，堵塞或损坏。使用一次性3毫升注射器，用无菌水填充管子，并将管子连接到一次性注射器上。然后将一块副泡沫放在生物安全柜的表面上，并向其中添加一池无菌水。将连接到针头的管道部分浸入无菌水池中。接下来，将一次性注射器从管口拉出，以防止任何空气在取出注射器时进入管路系统。然后用无菌水填充10微升汉密尔顿注射器。接下来，通过将汉密尔顿注射器的针尖浸入副膜上的无菌水池中，将汉密尔顿注射器针头连接到管路的开口端。按住泵屏幕上的快速反向按钮，将推杆块移动到注射器的近似长度。接下来，拧下支架夹紧旋钮以松开推杆和注射器支架块上的固定支架。随后，将汉密尔顿注射器装载到注射器支架块上，并按照制造商的说明固定注射器。要在泵设置屏幕中调整参数，请按“力”按钮并将力水平设置为 30%然后接受更改以返回设置屏幕。按“快速启动”按钮，选择“方法”，然后选择“仅注入”选项。对于注射器选项，选择汉密尔顿 1700，然后单击玻璃“并选择 10 微升。稍后，选择输液速率和注射量。按下“注入”按钮，从汉密尔顿注射器中弹出水，同时让管道和注射针充满水。接下来，通过将注射针头放入病毒原液的等分试样中来提取病毒。然后通过按下反向按钮稍微拉回汉密尔顿注射器，在管中引入一个小气泡。麻醉动物后，用镊子抓住结膜。然后将针头插入结膜，直到斜面完全被结膜覆盖，然后将针头放在眼球上。同时，通过使用脚踏开关按下“开始”按钮来开始注射。为了检查AAV载体的分布，用苏木精和伊红对眼部和周围组织进行染色。代表性矢状切片表明，印度墨水的分散主要发生在眼周松散结缔组织中巩膜外表面的眼外肌附近。为了确认注射后八周自辅AAV 8的转导，通过免疫荧光染色检查横截面中的绿色荧光蛋白丰度。结果表明，AAV 8载体有效地转导了眼睛和角膜后方的眼周肌肉。为了分析载体生物分布，使用转基因特异性引物和探针通过定量聚合酶链反应检查载体基因组拷贝数和其他器官（包括肝脏和心脏）中的不同眼室。定量逆转录聚合酶链反应证实了转基因的表达，这表明结膜下注射AAV 8导致眼睑、结膜、角膜和视神经中的转基因表达。气泡将作为管中的水和治疗药物之间的屏障。该技术允许其他研究人员探索结膜下给药途径用于眼科溶液和多种疾病的眼部基因治疗。

Research

JoVE Journal

Medicine

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

科研

医学

Protocol for Long Duration Whole Body Hyperthermia in Mice

持续时间较长的小鼠全身热疗协议

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

An Isolated Working Heart System for Large Animal Models

一个孤立的工作心脏系统的大型动物模型

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

小型动物正电子发射断层扫描/计算机断层扫描中的连续血液采样支持动脉输入函数的测量

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...