表1提供了该协议中使用的溶液和缓冲液的组成。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>溶液</td>
			<td colspan="2">组成</td>
		</tr><tr><td rowspan="4">AAV裂解缓冲液</td>
			<td colspan="2">1.2 mL 5 M NaCl 溶液</td>
		</tr><tr><td colspan="2">2 mL 1 M Tris-HCl pH 8.5 溶液</td>
		</tr><tr><td colspan="2">80 uL 1 M MgCl2 溶液</td>
		</tr><tr><td colspan="2">mQ 水至 40 mL</td>
		</tr><tr><td rowspan="3">AAV沉淀液</td>
			<td colspan="2">40克PEG 8000</td>
		</tr><tr><td colspan="2">50 mL 5 M NaCl 溶液</td>
		</tr><tr><td colspan="2">mQ 水至 100 mL</td>
		</tr><tr><td rowspan="5">15%碘克沙醇馏分</td>
			<td colspan="2">7.5 mL OptiPrep</td>
		</tr><tr><td colspan="2">3 mL 10X DPBS</td>
		</tr><tr><td colspan="2">6 mL 5 M NaCl 溶液</td>
		</tr><tr><td colspan="2">30 uL 1 M MgCl2 溶液</td>
		</tr><tr><td colspan="2">mQ 至 30 mL</td>
		</tr><tr><td rowspan="5">25%碘克沙醇馏分</td>
			<td colspan="2">12. 5 mL OptiPrep</td>
		</tr><tr><td colspan="2">3 mL 10X DPBS</td>
		</tr><tr><td colspan="2">30 uL 1 M MgCl2 溶液</td>
		</tr><tr><td colspan="2">60 uL酚红溶液</td>
		</tr><tr><td colspan="2">mQ 至 30 mL</td>
		</tr><tr><td rowspan="4">40%碘克沙醇馏分</td>
			<td colspan="2">33.3 mL OptiPrep</td>
		</tr><tr><td colspan="2">5 mL 10X DPBS</td>
		</tr><tr><td colspan="2">50 uL 1 M MgCl2 溶液</td>
		</tr><tr><td colspan="2">mQ 至 50 mL</td>
		</tr><tr><td rowspan="2">60%碘克沙醇馏分</td>
			<td colspan="2">50 mL OptiPrep</td>
		</tr><tr><td colspan="2">100 uL酚红溶液</td>
		</tr><tr><td rowspan="3">AAV缓冲溶液</td>
			<td colspan="2">8 mL 5 M NaCl</td>
		</tr><tr><td colspan="2">20 uL 10% Pluronic F-68</td>
		</tr><tr><td colspan="2">PBS 至 200 mL</td>
		</tr></tbody></table>表1：该协议中使用的溶液的溶液组成。
1. Expi293细胞的三重转染
<ol><li>以 5 x 105 个活细胞 （vc）/mL 的初始密度接种 Expi293 细胞（参见材料表）。</li>
	<li>让细胞在37°C下用8%CO2和125rpm的振荡速度孵育，直到它们达到3-5×106vc / mL的密度。使用台盼蓝排除和自动细胞计数仪监测细胞密度和活力（参见材料表）。</li>
	<li>当细胞达到所需的细胞密度时，通过添加新鲜培养基将它们分成 1 到 10 个，使其总体积是其原始体积的十倍。如有必要，将细胞转移到更大的烧瓶中。返回孵化期。</li>
	<li>继续如步骤1.2-1.3所述扩增细胞，直到它们在1L烧瓶中的250mL培养基中达到至少2×106 vc / mL的密度。</li>
	<li>转染前一天，在 250 mL 培养基中将细胞稀释至 2 x 106 vc/mL，必要时弃去多余的细胞。将细胞孵育过夜。</li>
	<li>在转染当天，将一半的细胞在18°C下以58× g 离心5分钟。 将细胞沉淀重悬于相同体积（125mL）的新鲜培养基中。细胞活力应接近98%。</li>
	<li>准备两个 50 mL 锥形管。将一个标记为“PEI”，另一个标记为“DNA”。</li>
	<li>在标有 PEI 的锥形管中，在 OptiMEM 培养基中将 1.2 mL PEI（聚乙烯亚胺，参见 材料表）稀释至总体积为 12.5 mL。</li>
	<li>在标记DNA的锥形管中，在OptiMEM培养基中制备质粒DNA的等摩尔溶液，以总体积为12.5mL的DNA获得总体积为500μg的DNA。</li>
	<li>将DNA溶液加入PEI溶液中，倒置数次充分结合，并在室温下孵育10分钟。 注意：至关重要的是，允许DNA-PEI溶液孵育整整10分钟，以使PEI与DNA形成带电复合物。</li>
	<li>DNA-PEI溶液孵育10分钟后，使用血清移液管将DNA-PEImax溶液缓慢施加到Expi293细胞上。将转染的细胞送回培养箱，让它们孵育72小时（图1）。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66460/66460fig01.jpg"> 图 1：转染 2 天后表达 GFP 的 Expi293 细胞。 用含有GFP基因的质粒转染后，Expi293细胞瞬时表达eGFP。细胞形态是圆形的。该图像的曝光时间为15 ms。使用配备落射荧光照明和 10x/0.30 物镜的倒置显微镜获取显微镜图像。比例尺 = 100 μm. <a href="https://www.jove.com/files/ftp_upload/66460/66460fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
2. rAAV载体纯化
<ol><li>72小时后，将悬浮细胞转移到两个250mL锥形管中，并在4°C下以415× g 旋转10分钟。</li>
	<li>将上清液培养基倒入新鲜的500mL锥形管中，并在0°C下储存在冰上以备后用。</li>
	<li>将每个细胞沉淀重悬于10mL AAV裂解缓冲液中（表1）。将两种裂解物集中在其中一个试管中。用另外 5 mL AAV 裂解缓冲液冲洗另一支试管，然后将其加入混合的裂解物中。在-70°C冷冻。 注意：此时可以暂停实验。将上清液培养基储存在-70°C而不是冰上。</li>
	<li>从步骤2.2中向上清液中加入1：4体积的AAV沉淀溶液。倒置几次以充分混合并在0°C下在冰上孵育至少2小时或过夜。</li>
	<li>将孵育溶液在4°C下以3000× g 离心1小时。</li>
	<li>弃去上清液，将含有病毒沉淀的沉淀重悬于5mL AAV裂解缓冲液中。</li>
	<li>在水浴中以37°C解冻细胞裂解物。</li>
	<li>将病毒沉淀与细胞裂解物混合。这是粗裂解物。用额外的 5 mL AAV 裂解缓冲液冲洗含有病毒沉淀的离心管，并与粗裂解物混合。</li>
	<li>将粗裂解物冷冻至-70°C，然后在37°C解冻。 再次重复此循环。 注意：粗裂解物在37°C下放置的时间不应超过解冻所需的时间。</li>
	<li>第三次解冻后，立即向粗裂解物中加入4μL苯并酶，倒置混合，并在37°C下孵育30分钟。</li>
	<li>在18°C下以650× g 离心粗裂解物10分钟。</li>
	<li>将上清液转移到干净的 50 mL 锥形管中。这是原始病毒。丢弃颗粒。</li>
	<li>在18°C下以3000× g 离心粗病毒30分钟以清除污染蛋白质。</li>
	<li>将上清液转移到干净的 50 mL 锥形管中。这是澄清的病毒。 注意：此时可以暂停实验。将澄清的病毒冷冻至-70°C。</li>
	<li>设置带有四个蠕动管的多通道蠕动泵（参见 材料表）。将毛细管连接到每根管的两端。</li>
	<li>将泵输入侧的毛细管放入装满去离子水的烧杯中。将泵输出侧的毛细管放入空烧杯中。以 25.0 rpm 的速度运行蠕动泵，用去离子 （DI） 水冲洗管道。</li>
	<li>彻底冲洗后，通过运行泵清空蠕动管，直到管子仅充满空气。将所有毛细管放在干净的无绒抹布上。</li>
	<li>将四个超速离心管放在泵输出侧的架子上。</li>
	<li>使用 10 mL 血清移液管小心地将 10 mL 澄清的病毒分配到每个超速离心管中。注意不要引入气泡。 注意：如有必要，每个试管中澄清的病毒体积可以拉伸至每管最多 12 mL。在下面的步骤2.25中适当减少60%碘克沙醇的量。</li>
	<li>使用蠕动泵将澄清的病毒从最低密度到最高密度的碘克沙醇级分（表1）覆盖。首先添加 15% 的分数，然后是 25% 的分数、40% 的分数，最后是 60% 的分数。将 22 mL（每个超速离心管 5.5 mL）的 15% 碘克沙醇部分转移到干净的 50 mL 锥形管中。将泵输入侧的毛细管放入管中，注意所有毛细管都接触管底。</li>
	<li>启动泵，让管道充满碘克沙醇馏分。当碘克沙醇馏分到达泵输出侧毛细管的末端时，停止泵。</li>
	<li>将一个输出毛细管插入每个装有澄清病毒的超速离心管中，注意毛细管接触每个超速离心管的底部。 注意： 毛细血管中没有空气是至关重要的。碘克沙醇可能以略有不同的速率流过蠕动管，因此在将其插入澄清的病毒之前，请确保每个单独的输出毛细管都完全充满碘克沙醇。可能需要多次停止和启动泵。</li>
	<li>启动泵并让超速离心管充满。当 15% 馏分中的最后一个即将被吸入输入毛细管时，停止泵。至关重要的是，没有气泡进入蠕动管。 注意： 如果气泡进入输入毛细管，泵可以以相反方向短暂运行以将它们推回。</li>
	<li>将 25% 碘克沙醇组分的 22 mL（每个超速离心管 5.5 mL）转移到 50 mL 锥形管中。注意所有毛细管都接触管子底部并启动泵。当 25% 馏分中的最后一个即将被吸入输入毛细管时，停止泵。</li>
	<li>用 20 mL（每根超速离心管 5 mL）的 40% 碘克沙醇馏分重复步骤 2.24，然后用 24 mL（每根超速离心管 6 mL）的 60% 馏分重复步骤 2.24。</li>
	<li>如果超速离心管中仍有未填充的体积，请继续添加更多的 60% 馏分，直到超速离心管完全充满液体。</li>
	<li>填充每个试管，直到裂解物在顶部形成圆顶，但不会从试管中溢出。停止泵并小心地去除每个输出毛细管，注意不要干扰碘克沙醇梯度。</li>
	<li>用垫片盖住每个超速离心管，并将其装入 70 Ti 型转子（参见 材料表）。 注意： 确保转子适当平衡。请勿在未经适当培训的情况下尝试操作超速离心机。</li>
	<li>将70型Ti转子加载到超速离心机中，并在18°C下以489,000× g 离心1小时。</li>
	<li>离心后，从超速离心机中卸下转子。使用尖嘴钳小心地从转子上取下每个超速离心管，注意不要干扰碘克沙醇梯度。</li>
	<li>使用带夹子的支撑环支架固定一个超速离心管。</li>
	<li>将 20 GA 针头连接到 5 mL 注射器并放在一边。使用无绒擦拭布从超速离心管上取下盖子。</li>
	<li>用针头穿透超速离心管壁，在40%-60%碘克沙醇界面下方约3毫米处（图2）。</li>
	<li>缓慢吸出界面和部分 40% 馏分。避免吸出 40% 馏分的顶部，总抽吸量约为 4 mL。</li>
	<li>将一根手指放在超速离心管的开口顶部，拉出注射器并将吸入的 AAV 馏分转移到 50 mL 锥形管中。将注射器丢弃在锐器容器中。丢弃超速离心管。</li>
	<li>对每个超速离心管重复步骤2.31-2.35。</li>
	<li>在AAV裂解缓冲液中将吸出的AAV馏分稀释约两倍至40mL的体积。</li>
	<li>按照步骤 2.16-2.17 中的说明冲洗蠕动管。 注意：此时可以暂停实验。将稀释的AAV馏分在0°C储存过夜。</li>
	<li>将稀释的 AAV 馏分各 20 mL 加载到两个新的超速离心管中。</li>
	<li>对于第二轮碘克沙醇密度梯度离心，如上所述，仅使用10 mL（每个超速离心管5 mL）的40%馏分和14 mL（每个超速离心管7 mL）的60%馏分，将稀释的AAV馏分铺在下面。重复步骤2.29-2.36进行超速离心和抽吸。 注意： 在存放蠕动泵和管道之前，请按照步骤 2.16-2.17 中的说明用去离子水冲洗管道。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66460/66460fig02.jpg"> 图 2：碘克沙醇梯度，标有 40%-60% 碘克沙醇界面。 （A）第一碘氧沙醇梯度。酚红用于 40% 碘克沙醇和 60% 碘克沙醇馏分。由于两个馏分之间的 pH 值不同，它显示为不同的颜色。箭头表示注射器应插入何处以收获 rAAV 馏分，就在 40%-60% 碘克沙醇界面下方。（B）第二碘氧醇梯度。此步骤仅使用40%和60%的碘克沙醇馏分。箭头表示注射器应插入何处以收获 rAAV 馏分。 <a href="https://www.jove.com/files/ftp_upload/66460/66460fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
3. 缓冲液更换和病毒浓缩
<ol><li>从第二轮碘克沙醇密度梯度离心中得到AAV馏分后，用AAV缓冲液稀释两倍。</li>
	<li>通过在过滤器顶部加入 20 mL AAV 缓冲溶液来平衡离心过滤器。在18°C下以3000× g 离心装置5分钟，并弃去流经。</li>
	<li>将稀释的AAV馏分加入离心过滤器中。在18°C下以3000× g 离心装置5分钟，并弃去流经。</li>
	<li>在过滤器顶部加入 20 mL AAV 缓冲溶液。在18°C下以3000× g 离心装置5分钟，并弃去流经。 注意：如果离心滤膜堵塞，导致溶液流出效率低下，请小心地使用 P200 微量移液器在过滤器顶部上下抽取溶液。请格外小心，不要用微量移液器吸头接触过滤器。</li>
	<li>再重复步骤 3.4 两次。 注意：通过在过滤器顶部保持至少 1 mL 的体积，确保 AAV 不会过度浓缩。可能需要调整离心时间以防止过度浓缩。如果AAV浓度过高，可能会发生降水。</li>
	<li>对于 1012 vg/mL 范围内的最终滴度，将病毒离心至最终体积约为 1 mL。 注意：根据所使用的血清型骨架和病毒的包装效率，您可能需要将病毒浓缩在较小的体积中。</li>
	<li>使用p1000微量移液器将浓缩的AAV从离心过滤器的顶部转移到微量离心管中。使用p200微量移液器用50μL AAV缓冲液洗涤离心过滤器，以收集任何残留的病毒并与其余的浓缩AAV汇集。</li>
	<li>留出 2 μL 等分的浓缩 AAV 进行滴度。请参阅 补充表1 ，查看可用于qPCR的ITR引物。</li>
	<li>将浓缩的AAV通过0.2μm注射器过滤器进行灭菌。 注意： 对小体积进行灭菌时，请注意选择直径较小的注射器过滤器，以尽量减少病毒损失。使用小直径的低蛋白结合过滤器将导致最小的病毒损失（数据未显示）。</li>
	<li>灭菌的AAV可以立即用于转导细胞或储存在4°C下，以便在4周内使用。应将其储存在-70°C下，以便长期储存。</li>
</ol>

通过双碘沙醇密度梯度离心分离 AAV

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作者没有披露任何信息。

双碘克沙醇密度梯度纯化方案是通用方法，因为它适用于任何AAV突变体变体，无论其受体特异性如何。早期的AAV纯化方法依赖于颗粒密度，包括CsCl中的等脁离心和连续蔗糖密度梯度离心19。后来，开发了血清型特异性方法，该方法利用与琼脂糖柱20 结合的单克隆抗体。1999 年开发了一种使用不连续碘克沙醇梯度的基于密度的新型纯化方法，并产生了比从 CsCl 梯?...

重组腺相关病毒 （rAAV） 载体是治疗遗传性疾病（包括脊髓性肌萎缩症、视网膜营养不良症和 A 型血友病）的广泛使用工具 1,2,3。rAAV 载体经过工程设计，缺乏野生型 AAV 4 中存在的病毒基因，野生型 AAV4 是一种具有线性单链 4.7 kb DNA 基因组的小型非包膜二十面体病毒。AAV 于 1960 年代首次被发现是腺病毒制剂的污染物5。尽管其衣壳尺寸小，将可包装的转基因的大小限制为最大 4.9 kb（不包括ITRs 6），但 AAV 可用于转基因递送，因为它在人类中无致病性，允许在许多分裂和非分裂细胞类型中表达转基因，并且免疫原性作用有限7。
作为依赖细小病毒属的成员，rAAV 的产生依赖于腺病毒或单纯疱疹病毒8 中存在的辅助基因的表达。已经开发了几种产生 rAAV 的策略，但在用腺病毒 E1A/E1B 辅助基因转化的 HEK293 细胞中生产是当今使用的最成熟的方法9。rAAV 生产的一般方法始于 HEK293 细胞的转染，其中三种质粒分别含有反转末端重复序列 （ITR）、AAV 代表 和 帽 基因以及其他腺病毒辅助基因中的转基因。转染后 72 小时，收获并处理细胞以纯化含有转基因的 rAAV。
在开发用于治疗目的的新型rAAV载体时，一个主要目标是生产具有更高转导效率的载体。靶细胞转导效率的提高意味着 rAAV 必要临床剂量的降低，从而降低从抗体介导的中和到急性毒性的不良免疫原性反应的可能性10,11。为了提高rAAV载体的转导功效，可以对包装的基因组或衣壳进行改变。通过包装基因组设计调整转导功效的可行方法包括掺入强效和组织特异性启动子、深思熟虑地选择 mRNA 加工元件以及编码序列优化以提高翻译效率12。对衣壳进行改变的目的是增加目标人类细胞类型的趋向性。开发新的 rAAV 转基因递送载体衣壳的努力通常以专注于合理设计具有靶向特定细胞受体的特定突变的 AAV 衣壳或定向进化以从高复杂性组合衣壳库中识别具有特定细胞类型嗜性的衣壳，而不靶向一种特定受体（尽管有些小组结合了这些方法）13，14,15.在定向进化方法中，组合衣壳文库是使用在衣壳外部具有突变可变区域的特定血清型骨架构建的 16。组合衣壳文库通常由非源自人类的 AAV 血清型构建，从而降低临床使用期间预先存在免疫的风险10。因此，可应用于任何血清型的纯化方法都是理想的选择，可以消除对作为这些文库骨架的不太常用的血清型进行血清型特异性优化的需要。
碘克沙醇密度梯度离心用于纯化高滴度的具有高感染性的 rAAV17。在该方案中，rAAV在悬浮细胞培养物中产生，以减少生产大滴度AAV所需的劳动力。还包括一个离心步骤，以澄清细胞裂解物，以减少污染蛋白质的存在并降低病毒沉淀的风险。该方案是一种具有成本效益的方法，用于生产适合临床前使用的高纯度rAAV制剂。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5810 R benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td></td>
		</tr>
		<tr>
			<td>8-channel peristaltic pump&#160;</td>
			<td>Watson-Marlow</td>
			<td>020.3708.00A</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter&#160;</td>
			<td>NanoEntek</td>
			<td>EVE-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-E high-speed centrifuge</td>
			<td>Beckman Coulter</td>
			<td>369001</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Thermo Scientific</td>
			<td>88701</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Labconco</td>
			<td>322491101</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator with shaker&#160;</td>
			<td></td>
			<td></td>
			<td>Set at 8% CO2 and 37 &#176;C</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339650</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Disposable micro-pipets</td>
			<td>Fisherbrand</td>
			<td>21-164-2G</td>
			<td>Capillaries</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline without CaCl2 and MgCl2&#160; (DPBS) (10x)</td>
			<td>Sigma-Aldrich</td>
			<td>D1408</td>
			<td></td>
		</tr>
		<tr>
			<td>ECLIPSE Ts2R-FL inverted microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expi293 Expression Medium</td>
			<td>Gibco</td>
			<td>A1435101</td>
			<td></td>
		</tr>
		<tr>
			<td>Expi293F cells</td>
			<td>Gibco</td>
			<td>A14527</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1126-7810</td>
			<td>1000 &#181;L</td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td>200 &#181;L</td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>Filter tips</td>
			<td>USA Scientific</td>
			<td>1121-3810</td>
			<td>10 &#181;L</td>
		</tr>
		<tr>
			<td>Hypodermic needles</td>
			<td>Tyco Healthcare</td>
			<td>820112</td>
			<td>20 GA x 1-1/2 A</td>
		</tr>
		<tr>
			<td>Ice bucket with lid</td>
			<td>VWR</td>
			<td>10146-184</td>
			<td></td>
		</tr>
		<tr>
			<td>JS-5.3 rotor</td>
			<td>Beckman Coulter</td>
			<td>368690</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride solution (1 M)</td>
			<td>Millipore Sigma</td>
			<td>M1028-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal stand and clamp&#160;</td>
			<td>Fisherbrand</td>
			<td>05-769-6Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22600028</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Needle nose pliers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced-Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep density gradient media (iodixanol)</td>
			<td>Serumwerk</td>
			<td>AXS-1114542</td>
			<td>60% iodixanol solution</td>
		</tr>
		<tr>
			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F144059M</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 Pipet</td>
			<td>Gilson</td>
			<td>F144054M</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 Pipet</td>
			<td>Gilson</td>
			<td>F144056M</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipet</td>
			<td>Gilson</td>
			<td>F144058M</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red&#160;solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4474</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid XP pipette controller</td>
			<td>Drummond Scientific</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid pCapsid</td>
			<td>De novo or Addgene, etc.&#160;</td>
			<td>N/A</td>
			<td>We used pACGrh74.&#160;</td>
		</tr>
		<tr>
			<td>Plasmid pHelper</td>
			<td>Addgene</td>
			<td>112867</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid pTransgene</td>
			<td>De novo or Addgene, etc.&#160;</td>
			<td>N/A</td>
			<td>We used pdsAAV-GFP.</td>
		</tr>
		<tr>
			<td>Pluronic F-68 polyol solution (10%)</td>
			<td>Mp Biomedicals</td>
			<td>92750049</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol 8000</td>
			<td>Research Products International</td>
			<td>P48080-500.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine HCl Max (PEI-Max)</td>
			<td>Polysciences</td>
			<td>NC1038561</td>
			<td>Dilute in water to 40 &#956;M</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubes, sterile</td>
			<td>Corning</td>
			<td>431123</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubes, sterile</td>
			<td>Corning</td>
			<td>430776</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Polypropylene Optiseal tubes</td>
			<td>Beckman Coulter</td>
			<td>361625</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP250-B</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP225-B</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP210-B</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Serological pipettes</td>
			<td>Alkali Scientific</td>
			<td>SP205-B</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV1000</td>
			<td>1 L</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV50-0</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV250</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Shaker flasks</td>
			<td>Fisherbrand</td>
			<td>PBV12-5</td>
			<td>125 mL</td>
		</tr>
		<tr>
			<td>Sodium chloride solution (5 M)</td>
			<td>Fisher Scientific</td>
			<td>NC1752640</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringes</td>
			<td>Fisherbrand</td>
			<td>14-955-458</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore</td>
			<td>SLGV013SL</td>
			<td>0.22 micron</td>
		</tr>
		<tr>
			<td>Tris-HCl pH 8.5 (1 M)</td>
			<td>Kd Medical</td>
			<td>RGE3363</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rack assembly</td>
			<td>Beckman Coulter</td>
			<td>361646</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube spacers (x4)</td>
			<td>Beckman Coulter</td>
			<td>361669</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing for peristaltic pump</td>
			<td>Fisher Scientific</td>
			<td>14190516</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 70 Ti fixed-angle titanium rotor</td>
			<td>Beckman Coulter</td>
			<td>337922</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra low temperature freezer</td>
			<td></td>
			<td></td>
			<td>Set at -70 &#176;C</td>
		</tr>
		<tr>
			<td>Vivaspin 20 centrifugal concentrator</td>
			<td>Sartorius</td>
			<td>VS2041</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath&#160;</td>
			<td></td>
			<td></td>
			<td>Set at 37 &#176;C</td>
		</tr>
	</tbody>
</table>

suspension culture production and purification of adeno-associated virus by iodixanol density gradient centrifugation for in vivo applications

该协议描述了通过碘克沙醇密度梯度离心产生和纯化的重组腺相关病毒（rAAV），这是一种与血清型无关的纯化AAV的方法，于1999年首次描述。rAAV载体广泛用于基因治疗应用，将转基因递送至各种人类细胞类型。在这项工作中，重组病毒是通过用编码转基因、载体衣壳和腺病毒辅助基因的质粒在悬浮培养物中转染 Expi293 细胞来生产的。碘克沙醇密度梯度离心法根据颗粒密度纯化完整的AAV颗粒。此外，这种现在无处不在的方法还包括三个步骤，以提高病毒总产量，降低由于污染蛋白质而沉淀的风险，并进一步浓缩最终病毒产物，分别：使用聚乙二醇（PEG）和氯化钠溶液从细胞培养基中沉淀病毒颗粒，引入第二轮碘克沙醇密度梯度离心， 以及通过离心过滤器进行缓冲液置换。使用这种方法，可以在体内使用时始终如一地达到 1012 个病毒颗粒/mL 的异常纯度。

Recombinant adeno-associated viral (rAAV) vectors are widely used tools for the treatment of genetic diseases, including spinal muscular atrophy, retinal dystrophy, and hemophilia A1,2,3. rAAV vectors are engineered to lack viral genes present in wild-type AAV4, a small, non-envelope icosahedral virus with a linear single-stranded 4.7 kb DNA genome. AAV was first discovered in the 1960s as a contaminant of adenovirus preparations5. Despite its small capsid size, which limits the size of the transgene that can be packaged to a maximum of 4.9 kb excluding ITRs6, AAV is useful for transgene delivery because it is non-pathogenic in humans, allows expression of transgene in many dividing and nondividing cell types, and has limited immunogenic effects7.
As members of the genus dependoparvovirus, the production of rAAVs relies on the expression of helper genes present in adenovirus or herpes simplex virus8. Several strategies to produce rAAV have been developed, but production in HEK293 cells transformed with the adenoviral E1A/E1B helper genes is the most established method used today9. The general approach of rAAV production begins with the transfection of HEK293 cells with three plasmids containing the transgene within inverted terminal repeats (ITRs), AAV rep and cap genes, and additional adenoviral helper genes, respectively. Seventy-two hours after transfection, cells are harvested and processed to purify rAAV containing the transgene.
In the development of new rAAV vectors for therapeutic purposes, a major goal is producing vectors with increased transduction efficiency. An increase in the transduction efficiency of target cells would mean a decrease in the necessary clinical dose of rAAV, thus decreasing the likelihood of adverse immunogenic effects ranging from antibody-mediated neutralization to acute toxicities10,11. To improve the transduction efficacy of rAAV vectors, alterations can be made to the packaged genome or to the capsid. Viable methods to tune transduction efficacy via packaged genome design include the incorporation of strong and tissue-specific promoters, thoughtful selection of mRNA processing elements, and coding sequence optimization to improve translation efficiency12. Alterations to the capsid are made with the goal of increasing tropism for target human cell types. Efforts towards developing new rAAV transgene delivery vector capsids are generally characterized by a focus on either rational design of AAV capsids with specific mutations targeting specific cell receptors or directed evolution to identify capsids with tropism for specific cell types from high-complexity combinatorial capsid libraries without targeting one specific receptor (although some groups combine these approaches)13,14,15. In the directed evolution approach, combinatorial capsid libraries are constructed using a particular serotype backbone with mutated variable regions on the capsid exterior16. Combinatorial capsid libraries are often constructed from AAV serotypes not originating in humans, decreasing the risk of preexisting immunity during clinical use10. Therefore, purification methods that can be applied to any serotype are ideal to eliminate the need for serotype-specific optimization for the less commonly used serotypes serving as backbones for these libraries.
Iodixanol density gradient centrifugation is utilized to purify high titers of rAAV with high infectivity17. In this protocol, rAAV is produced in suspension cell culture to decrease the amount of labor needed to produce large titers of AAV. A centrifugation step is also included to clear cell lysate to reduce the presence of contaminating proteins and decrease the risk of virus precipitation. This protocol is a cost-effective method to produce preparations of high-purity rAAV suitable for pre-clinical use.

作者聚焦：用于临床前应用的高效腺相关病毒分离

悬浮培养物生产和纯化腺相关病毒的碘克沙醇密度梯度离心用于体内应用

We use a directed evolution approach to identify adeno-associated viral capits with higher tropism for target human cell types. Through this research, we aim to produce recombinant adeno-associated viral vectors with improved target cell transduction and clinical relevance within the field of gene therapy. Our group recently de-targeted several AAV serotypes from the liver by incorporating capsid residues original to an AAV serotype that naturally possesses decreased liver tropism.If successfully expanded to additional serotypes, this provides the groundwork to decrease necessary RAAV clinical dose by peripheral injection and reduce adverse patient outcomes. The IODIXanol purification method while being cost effective is difficult to scale up. Additionally, in downstream applications, the directed evolution approach is performed with animal models or in vitro.So capsid isolated by directed evolution may not transduce in vivo targets in humans to the same extent as in preclinical models. This protocol is affordable and accessible for smaller labs. Additionally, this method yields high purity, recombinant adeno-associated virus that can be used for downstream preclinical in vivo applications.

该方法可用于获得每mL至少10-12个病毒颗粒的滴度。使用补充表1中提供的ITR引物通过qPCR获得滴度（图3），通过ddPCR或任何其他滴度方法。使用包装效率低下的衣壳的帽基因可能导致滴度欠佳。
结果欠佳的另一个可能来源是 Expi293 细胞的转导效率差。建议在转染当天的细胞密度为 3-4 x 106 vc/mL，并且细胞活力接近 98%。在...

Watch this Scientific Journal Video about Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications at JoVE.com

腺相关病毒在悬浮细胞培养物中产生，并通过双碘克沙醇密度梯度离心纯化。包括提高总病毒产量、降低病毒沉淀风险和进一步浓缩最终病毒产物的步骤。预期最终滴度达到 1012 个病毒颗粒/mL，适用于临床前 体内 使用。

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a ...

我们使用定向进化方法来鉴定对靶人类细胞类型具有更高趋向性的腺相关病毒帽。通过这项研究，我们的目标是生产重组腺相关病毒载体，在基因治疗领域具有更好的靶细胞转导和临床相关性。我们的小组最近通过掺入天然具有降低肝脏嗜性的AAV血清型的原始衣壳残基，从肝脏中去靶向了几种AAV血清型。如果成功扩展到其他血清型，这将为通过外周注射减少必要的 RAAV 临床剂量和减少患者不良结局奠定基础。碘二醇纯化方法虽然具有成本效益，但很难扩大规模。此外，在下游应用中，定向进化方法是使用动物模型或体外进行的。因此，通过定向进化分离的衣壳在人体中的转导程度可能与临床前模型相同。该协议价格实惠，适用于小型实验室。此外，该方法可产生高纯度的重组腺相关病毒，可用于下游临床前体内应用。

suspension-culture-production-purification-adeno-associated-virus

Research

JoVE Journal

Medicine

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

1.4K 次观看.  University of Florida. 我们使用定向进化方法来鉴定对靶人类细胞类型具有更高趋向性的腺相关病毒帽。通过这项研究，我们的目标是生产重组腺相关病毒载体，在基因治疗领域具有更好的靶细胞转导和临床相关性。我们的小组最近通过掺入天然具有降低肝脏嗜性的AAV血清型的原始衣壳残基，从肝脏中去靶向了几种AAV血清型。如果成功扩展到其他血清型，这将为通过外周注射减少必要的 RAAV ?...