我们感谢科英布拉临床和生物医学研究所 （iCBR） 和创新生物医学与生物技术中心 （CIBB） 的 Mónica Zuzarte 博士在 rAAV 的 TEM 分析方面提供的合作、见解和技术援助。我们感谢科英布拉大学神经科学和细胞生物学中心（CNC-UC）和科英布拉大学跨学科研究所（IIIUC）的Dominique Fernandes博士，感谢她关于初级神经元培养实验的宝贵技术援助和见解。本研究所必需的 pRV1、pH21 和 pFdelta6 质粒由阿伯丁大学生命科学与医学院医学院的 Christina McClure 博士慷慨提供，我们对此表示感谢。这项工作由欧洲区域发展基金 （ERDF） 通过 Centro 2020 区域运营计划资助;通过COMPETE 2020--竞争力和国际化业务计划，以及通过FCT - Fundação para a Ciência e a Tecnologia的葡萄牙国家基金，在以下项目下：UIDB/04539/2020、UIDP/04539/2020、LA/P/0058/2020、ViraVector（CENTRO-01-0145-FEDER-022095）、Imagene （PTDC/BBB-NAN/0932/2014 |POCI-01-0145-FEDER-016807）、重置 - IDT-COP （CENTRO-01-0247-FEDER-070162）、抗击 Sars-CoV-2 （CENTRO-01-01D2-FEDER-000002）、BDforMJD （CENTRO-01-0145-FEDER-181240）、ModelPolyQ2.0 （CENTRO-01-0145-FEDER-181258）、MJDEDIT （CENTRO-01-0145-FEDER-181266）;由美国葡萄牙生物医学研究基金 （APBRF） 和 Richard Chin 和 Lily Lock Machado-Joseph 疾病研究基金、ARDAT 根据 IMI2 JU 赠款协议第 945473 号，由欧盟和 EFPIA 支持;GeneT-Teaming Project 101059981欧盟的 Horizon Europe 计划提供支持。M.M.L.得到了 2021.05776.BD 的支持;C.H. 得到了 2021.06939.BD 的支持;A.C.S.得到了 2020.07721.BD 的支持;D.D.L.得到了 2020.09668.BD 的支持。 图1 是使用 BioRender.com 创建的。

通过单步肝素亲和层析纯化 AAV 载体

作者声明没有利益冲突。

快速扩展的AAV载体工具包已成为通过不同给药途径为多种细胞类型提供的最有前途的基因递送系统之一。在这项工作中，我们旨在开发一种改进的方案，用于花叶 rAAV 载体的生产、纯化和表征，以证明它们在临床前研究中的价值。为此，本文描述了 rAAV1/2 和 rAAV2/9 镶嵌载体的生成，但该过程也可用于纯化标准 rAAV2 载体（数据未显示）。
镶嵌 rAAV 是按照使用 PEI 作为转染试剂的优化转染方法生产的。选择瞬时转染方法是因为其具有更大的灵活性和速度，在早期临床前研究中具有相当大的优势。一旦特定转基因和血清型得到验证，就可以对生产系统进行微调，通过建立稳定的转染细胞系来实现更好的可扩展性和成本效益，该细胞系表达特定 Rep/Cap 基因的一个子集，以及感染过程提供的其他基因24。与磷酸钙转染相比，PEI具有几个优点。它是一种稳定且经济高效的转染试剂，可在更宽的 pH 范围内有效运行。此外，它还消除了转染后更换细胞培养基的需要，从而显著降低了成本和工作量69。
为了规避 CsCl 或碘克沙醇梯度带来的一些限制，通过亲和色谱法收获和纯化产生的 rAAV。该策略提供了一种简化且可扩展的方法，无需超速离心和梯度即可执行，从而产生干净且高的病毒滴度。事实上，使用FPLC系统的色谱技术可以通过在具有更高柱床高度的色谱柱中填充更多的填料体积来实现自动化和放大。本文中描述的方案可以很容易地适应以纳入 5 mL HiTrap 肝素 HP 柱（数据未显示）。此外，肝素柱可以多次重复使用，从而有助于提高该方法的成本效益。
然后对纯化的rAAVs进行滴度、纯度、形态特征和生物活性的表征。有趣的是，在考马斯蓝染色中，除了三种典型的病毒衣壳蛋白外，在 F8-F16 级分中还检测到了大约 17 kDa 的条带。然而，在rAAV的浓缩步骤之后，该条带不再存在。此外，在 B1 和 A69 标记时也可以检测到一些分子量低于 VP3 （<62 kDa） 的微弱条带，表明这些可能是 VP1、VP2 和 VP3 衣壳蛋白的片段70。另一种可能性是，这些实际上是其他共纯化的蛋白质，例如铁蛋白或其他具有多肽的细胞蛋白，这些多肽与AAV衣壳蛋白具有相似的蛋白质指纹图谱，并且可能参与AAV生物学，如前所述26,71,72。
透射电镜和电击器分析还揭示了不同产品中不同水平的空颗粒的存在。同样，其他研究先前报告了通过转染或感染方法制备的 rAAV 产生可变和高水平的 （>65%） 空衣壳24,73。rAAV 产生的机制始于新合成的 VP 蛋白快速形成空衣壳，然后是基因组包装到由 Rep 蛋白介导的预形成衣壳中的缓慢限速步骤74,75。因此，在rAAV生产中会产生空衣壳，尽管空衣壳和完整衣壳的比例可能因目标转基因的大小和序列以及细胞培养条件而变化58,73。空衣壳引起了一些担忧，因为由于缺乏感兴趣的基因组，它们无法提供治疗效果，并且还可能增加先天性或适应性免疫反应。然而，一些报告还表明，通过调整其比例，空的 AAV 衣壳可以作为 AAV 特异性中和抗体的高效诱饵，因此可以提高转导效率 60,76,77。如果空衣壳的存在是非常有害的，并且考虑到空颗粒的阴离子特性与全载体相比略少，则可能的解决方案可能涉及使用阴离子交换色谱技术进行第二次精纯纯化步骤64。
这项研究还提供了令人信服的证据，表明生成的镶嵌 rAAV 不仅能够有效地转导体 外 神经元培养物，而且在颅内注射 rAAV1/2 时也能有效地转导中枢神经系统。总体而言，这些结果表明，所描述的生产和纯化方案可在 6 天内获得高纯度和生物活性的 rAAV，从而在临床前研究中成为生成 rAAV 的通用且具有成本效益的方法。

腺相关病毒（AAV）载体正在成为当前基因治疗研究中最有前途的递送系统之一。AAV 最初于 19651 年被发现，是一种小型无包膜病毒，其二十面体蛋白衣壳直径约 25 nm，含有单链 DNA 基因组。AAV 属于细小病毒科和依赖细小病毒属，因为它们独特地依赖于与辅助病毒（如单纯疱疹病毒或更常见的腺病毒）共同感染以完成其裂解周期 2,3。
AAV 的 4.7 千碱基基因组由两个开放阅读框 （ORF） 组成，两侧是两个倒置末端重复序列 （ITR），形成特征性的 T 形发夹末端4。ITR 是唯一对 AAV 包装、复制和整合至关重要的顺式作用元件，因此是重组 AAV （rAAV） 载体中唯一保留的 AAV 序列。在该系统中，载体生产所需的基因以反式形式单独提供，允许将目的基因包装在病毒衣壳内 5,6。
每个病毒基因通过选择性剪接和起始密码子编码不同的蛋白质。在 Rep ORF 中，编码四种非结构蛋白（Rep40、Rep52、Rep68 和 Rep78），在病毒 DNA 的复制、位点特异性整合和包封中起着至关重要的作用 7,8。Cap ORF 用作在其 N 末端（VP1、VP2 和 VP3）表达彼此不同的三种结构蛋白的模板，这些结构蛋白以 1：1：10 的比例组装形成 60 聚体病毒衣壳 4,9。此外，嵌套在 Cap 基因中的替代 ORF 具有非常规 CUG 起始密码子，编码组装激活蛋白 （AAP）。这种核蛋白已被证明与新合成的衣壳蛋白 VP1-3 相互作用并促进衣壳组装10,11。
衣壳氨基酸序列的差异导致了 11 种天然存在的 AAV 血清型和从人类和非人类灵长类动物组织中分离出的 100 多种变体 7,12,13。结构可变区域构象的变化决定了来自不同菌株的衣壳的不同抗原特性和受体结合特异性。这导致不同哺乳动物器官的不同组织趋向性和转导效率14.
rAAV的早期生产方法依赖于腺病毒感染作为辅助目的15,16,17,18,19。尽管这种感染效率高且通常易于大规模生产，但这种感染仍会出现一些问题。即使在纯化和热变性步骤灭活之后，腺病毒颗粒仍可能存在于AAV制剂中，构成不必要的安全性问题20。此外，变性腺病毒蛋白的存在对于临床使用是不可接受的。其他生产策略利用重组单纯疱疹病毒株，这些病毒株经过工程改造，可将 Rep/Cap 和转基因带入靶细胞21 或杆状病毒-昆虫细胞系统22。尽管这些系统在可扩展性和GMP兼容性方面具有优势，但它们仍然面临类似的问题。
用于 rAAV 生产的三重转染方法已被普遍采用，以轻松克服这些问题。简而言之，rAAV 组装依赖于具有三个质粒的细胞的瞬时转染，这些质粒编码：1） 来自野生型 AAV2 基因组 （pITR） 的 ITR 之间的转基因表达盒;2） 复制和病毒粒子组装所需的 Rep/Cap 序列 （pAAV-RC）;3）最小的腺病毒蛋白（E1A、E1B、E4和E2A）以及辅助效应（pHelper）所需的腺病毒病毒相关RNA（6,20,23）。虽然质粒转染方法在临床前研究中为rAAV生产提供了简单性和灵活性，但当应用于大规模生产时，这些程序在可扩展性和可重复性方面存在局限性。作为一种替代方法，rAAV 生产可以通过使用 AAV 生产细胞系（贴壁和悬浮生长）来实现，稳定表达 AAV Rep/Cap 基因或 Rep/Cap 与载体构建体结合。在这些系统中，腺病毒辅助基因是通过质粒转染引入的。尽管这种策略提高了细胞培养过程的可扩展性，但它在技术上既复杂又耗时 21,24,25。
在任何一种情况下，生产细胞随后被裂解并进行一个或多个纯化步骤。目前，纯化rAAVs的主要方法包括使用氯化铯（CsCl）或碘克沙醇进行超高速密度梯度离心，然后或不使用色谱技术26。原始的病毒沉淀纯化方案使用硫酸铵，然后通过CsCl梯度进行两轮或三轮超速离心。该工艺的主要优点包括可以纯化所有血清型，并且能够根据其不同的密度从空衣壳中物理分离全颗粒。然而，这种方法复杂、耗时且可扩展性有限，往往导致产量低和样品质量低下 27,28,29,30。此外，由于 CsCl 可能对哺乳动物产生毒性作用，因此在体内研究之前通常需要对生理缓冲液进行透析。
碘克沙醇还被用作替代等渗梯度培养基来纯化 rAAV 载体，从安全性和载体效力的角度来看，碘克沙醇比 CsCl 更具优势。然而，与 CsCl 一样，碘克沙醇方法存在一些与细胞培养裂解物的负载能力相关的缺点（因此也存在 rAAV 纯化的可扩展性），并且它仍然是一种耗时且昂贵的方法30,31。
为了克服这些限制，研究人员将注意力转向了色谱技术。在这方面，已经开发了几种纯化方法，这些方法要么结合了亲和、疏水或离子交换色谱方法。这些方法依赖于特定血清型的生化特性，包括它们的天然受体，或病毒颗粒的电荷特性32。例如，AAV2、AAV3、AAV6 和 AAV13 优选与硫酸乙酰肝素蛋白聚糖 （HSPG） 结合的发现，为在亲和层析纯化中使用密切相关的肝素提供了可能性。然而，与HSPG的结合位点可能因血清型而异，以不同的方式介导AAV附着和靶细胞的感染2,33,34,35,36。另一方面，AAV1、AAV5 和 AAV6 与 N-连接的唾液酸 （SA） 结合，而 AAV4 使用 O 连接的 SA 2,14,34。遵循相同的原理，还基于粘蛋白（一种在 SA37 中高度富集的哺乳动物蛋白质）的使用开发了用于纯化 rAAV5 的单步亲和色谱方案。与基于肝素的技术一样，这种纯化也取决于所产生的特定血清型。除肝素和粘蛋白外，还探索了其他配体用于亲和层析，例如 A20 单克隆抗体和骆驼类单域抗体（AVB Sepharose 和 POROS CaptureSelect）22,23,38,39,40,41。改进先前现有纯化方法的其他创新策略包括在 rAAV 衣壳中引入小修饰以呈现特定的结合表位。例如，可以使用靶向这些表位的配体（分别为次氮基三乙酸镍和亲和素树脂）纯化六组氨酸标记或生物素化的 rAAV 42,43,44。
为了扩大rAAVs的预期特性，研究人员通过混合它们的衣壳来杂装病毒粒子。这是通过在生产过程中以等摩尔或不同比例提供来自两种不同AAV血清型的衣壳基因来实现的，从而产生由来自不同血清型的衣壳亚基混合物组成的衣壳结构34,45,46,47,48,49,50.先前的研究提供了物理证据，表明 AAV2 与 AAV1（1：1 比例）和 AAV2 与 AAV9（1：1 比例）共表达衣壳蛋白可分别产生镶嵌 rAAV1/2 和 rAAV2/9 载体45、46、48。产生花叶 rAAV 的一个主要好处是能够整合来自不同 AAV 血清型的有利性状，从而协同改善转基因表达和趋向性，同时保持 rAAV 生产过程中有用的其他特性。有趣的是，某些花叶变体甚至表现出与亲本病毒不同的新特性 46,47,49。通过利用 AAV2 的肝素结合能力，通过将 AAV2 与定向进化和/或合理设计产生的其他天然或新 AAV 衣壳混合，有可能产生和纯化镶嵌 rAAV 载体。尽管如此，以前的研究强调了在尝试组装镶嵌载体时衣壳亚基兼容性的重要性。例如，Rabinowitz 及其同事证明，尽管 AAV1、AAV2 和 AAV3 的转囊化导致了花叶衣壳的有效共组装，但这些血清型与 AAV4 的杂交阻碍了稳定病毒粒子的产生 34,45,47。此外，AAV1、AAV2 和 AAV3 与 AAV5 的相容性较低，因为以不同比例混合这些衣壳时获得的病毒滴度降低。有趣的是，镶嵌 rAAV2/5 显示出肝素结合特性降低，同时保持了与亲本 AAV5 一样的粘蛋白结合能力。然而，rAAV3/5 以 3：1 的比例保留了与肝素和粘蛋白的双重结合。总体而言，具有增强转导、特异性趋向性或低免疫原性的新镶嵌 rAAV 的生成可以从我们对衣壳组装和受体相互作用的理解中受益匪浅，而特定组合仍需要深入的研究和优化。
在本工作中，我们描述了使用优化的肝素亲和色谱法生产和纯化rAAVs的分步方案。rAAV 通过瞬时转染产生，并使用快蛋白液相色谱 （FPLC） 系统进行纯化。在对选定的纯化馏分进行浓缩后，所得病毒储备液在体外和体内的滴度、纯度、特性物理性质和生物活性方面进行了表征。作为概念验证，我们展示了该协议在生成镶嵌 rAAV1/2 和 rAAV2/9 载体方面的改进和适用性。每种血清型的选择都是基于它们截然不同的趋向性，这可能也赋予了马赛克版本独特的特征。AAV 血清型 1 对中枢神经系统 （CNS） 具有总体中等趋向性，具有转导神经元和神经胶质细胞的能力（在较小程度上），并在体内顺行和逆行方向进行轴突转运 2,7,8。此外，AAV 血清型 9 因其在新生儿和成年小鼠中具有穿过血脑屏障并靶向中枢神经系统的自然能力51,52。最后，鉴于 AAV 血清型 2 能够与肝素结合，因此选择其亲和层析33。纯化的 rAAV1/2 和 rAAV2/9 颗粒结合了两种亲本 AAV 血清型的特性，因此构成了 CNS 转导的合适载体 45,46,48,49。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% povidone-iodine</td>
			<td>Medline</td>
			<td>MDS093943</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Thermo Scientific</td>
			<td>11889684</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>VWR</td>
			<td>734-2325</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Stunner plate</td>
			<td>Unchained Labs</td>
			<td>701-2025</td>
			<td>96-well quantification plate for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples</td>
		</tr>
		<tr>
			<td>AAVpro Titration Kit (for Real-Time PCR) Ver.2</td>
			<td>Takara</td>
			<td>6233</td>
			<td>For determining the titer of AAV using RT-qPCR. This kit contains DNase I, Lysis Buffer, Dilution Buffer, positive control, Taq II mix, primer forward, primer reverse, water</td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Fisher Chemical</td>
			<td>A/0360/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>&#196;KTA pure 25</td>
			<td>Cytiva</td>
			<td>29018224</td>
			<td>FPLC system controlled by UNICORN software, version 6.3&#160;</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase-linked goat anti-mouse</td>
			<td>Invitrogen</td>
			<td>31328</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra-0.5 centrifugal filter unit</td>
			<td>Merck Millipore</td>
			<td>UFC5100</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon ultra-15 centrifugal filter unit</td>
			<td>Merck Millipore</td>
			<td>UFC9100</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Merck Millipore</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR detection system</td>
			<td>Biorad</td>
			<td>184-5096</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch Imaging System</td>
			<td>Bio-Rad Laboratories</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken polyclonal anti-GFP primary antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue G250</td>
			<td>Fisher Chemical</td>
			<td>C/P541/46</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Fisher Bioreagents</td>
			<td>BP17225</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>ECF Substrate for Western Blotting</td>
			<td>Cytiva</td>
			<td>RPN5785</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest SmaI</td>
			<td>Thermo Scientific</td>
			<td>FD0663</td>
			<td></td>
		</tr>
		<tr>
			<td>FEI-Tecnai G2 Spirit Biotwin</td>
			<td>FEI</td>
			<td>Biotwin</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biowest</td>
			<td>S1810</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence mounting medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar-carbon coated 200 mesh grid</td>
			<td>&#160;TAAB Laboratories Equipment</td>
			<td>F077/025</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas evacuation apparatus</td>
			<td>RWD</td>
			<td>R546W</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>10021083</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-chicken antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton needle 30G, Small Hub RN Needle, 25 mm, PST3</td>
			<td>Hamilton</td>
			<td>7803-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe (10 &#181;L)</td>
			<td>Hamilton</td>
			<td>7653-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP 1 x 5 mL</td>
			<td>Cytiva</td>
			<td>17040701</td>
			<td>Pre-packed heparin column</td>
		</tr>
		<tr>
			<td>HiTrap Heparin HP 5 x 1 mL</td>
			<td>Cytiva</td>
			<td>17040601</td>
			<td>Pre-packed heparin column</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Braun</td>
			<td>469860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Dechra Pharmaceuticals</td>
			<td>N/A</td>
			<td>Nimatek</td>
		</tr>
		<tr>
			<td>Low-retention microcentrifuge tubes (2 mL)</td>
			<td>Fisher Scientific</td>
			<td>11906965</td>
			<td></td>
		</tr>
		<tr>
			<td>Lunatic &#38; Stunner Client software</td>
			<td>Unchained Labs</td>
			<td>N/A</td>
			<td>Client analysis software version 8.0.1.235. Software for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Chemical</td>
			<td>M/4000/FP21</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-AAV, VP1, VP2 antibody (A69)</td>
			<td>American Research Products</td>
			<td>03-61057</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-AAV, VP1, VP2, VP3 antibody (B1)</td>
			<td>American Research Products</td>
			<td>03-61058</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuro2a</td>
			<td>American Type Culture Collection</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Gibco</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi EF</td>
			<td>Macherey-Nagel</td>
			<td>12738422</td>
			<td></td>
		</tr>
		<tr>
			<td>NZYColour Protein Marker II</td>
			<td>NZYtech</td>
			<td>MB09002</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-CMV-scGFP</td>
			<td>Addgene</td>
			<td>32396</td>
			<td>Addgene plasmid # 32396; http://n2t.net/addgene:32396; RRID:Addgene_32396</td>
		</tr>
		<tr>
			<td>pAAV-CMV-ssGFP</td>
			<td>Addgene</td>
			<td>105530</td>
			<td>Addgene plasmid # 105530; http://n2t.net/addgene:105530; RRID:Addgene_105530</td>
		</tr>
		<tr>
			<td>pAAV2/9n</td>
			<td>Addgene</td>
			<td>112865</td>
			<td>Addgene plasmid # 112865; http://n2t.net/addgene:112865; RRID:Addgene_112865</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>10342243</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher BioReagents</td>
			<td>BP2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-68 Non-ionic Surfactant (100x)</td>
			<td>Gibco</td>
			<td>24040032</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine MAX, MW 40,000</td>
			<td>Polysciences Europe</td>
			<td>24765</td>
			<td></td>
		</tr>
		<tr>
			<td>R500 Series Compact Small Animal Anesthesia Machine - Isoflurane</td>
			<td>RWD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Inlet Valve V9-IS</td>
			<td>Cytiva</td>
			<td>29027746</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample pump P9-S</td>
			<td>Cytiva</td>
			<td>29027745</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>10428420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Bioreagents</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PVDF syringe filter</td>
			<td>Fisher Scientific</td>
			<td>15191499</td>
			<td></td>
		</tr>
		<tr>
			<td>Stunner Platform</td>
			<td>Unchained Labs</td>
			<td>700-2002</td>
			<td>Equipment for the consecutive ultraviolet-visible light absorption, static light scattering, and dynamic light scattering analysis of rAAV samples</td>
		</tr>
		<tr>
			<td>Superloop 150 mL</td>
			<td>Cytiva</td>
			<td>18-1023-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Superloop 50 mL</td>
			<td>Cytiva</td>
			<td>18-1113-82</td>
			<td></td>
		</tr>
		<tr>
			<td>SURE 2 supercompetent cells</td>
			<td>Stratagene, Agilent Technologies</td>
			<td>HPA200152</td>
			<td></td>
		</tr>
		<tr>
			<td>Treated culture dishes</td>
			<td>Corning</td>
			<td>734-1711</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher BioReagents</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP153</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor 633</td>
			<td>Invitrogen</td>
			<td>W21404</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Dechra Pharmaceuticals</td>
			<td>N/A</td>
			<td>Sedaxylan</td>
		</tr>
		<tr>
			<td>Zeiss Axio Observer Z1</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>N/A</td>
			<td>Inverted fluorescence microscope equiped with an EC Plan-Neofluar 10x/0.30 objective and a Plan-Apochromat 40x/0.95 objective</td>
		</tr>
		<tr>
			<td>Zeiss Axio Scan.Z1</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>N/A</td>
			<td>Slide scanner fluorescence microscope equipped with a Plan-Apochromat 20x/0.8 objective</td>
		</tr>
		<tr>
			<td>Zeiss LSM 710</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td>N/A</td>
			<td>Inverted confocal microscope equipped with a Plan-Apochromat 40x/1.4 Oil DIC objective</td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 well Ibidi</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>8-well chamber slide</td>
		</tr>
	</tbody>
</table>

isolation of adeno-associated viral vectors through a single-step and semi-automated heparin affinity chromatography protocol

腺相关病毒（AAV）已成为一种越来越有价值的 体内 基因递送载体，目前正在进行人体临床试验。然而，纯化AAV的常用方法利用氯化铯或碘克沙醇密度梯度超速离心。尽管这些方法具有优点，但这些方法非常耗时，可扩展性有限，并且通常会导致纯度低的载体。为了克服这些限制，研究人员正在将注意力转向色谱技术。在这里，我们提出了一种优化的基于肝素的亲和层析方案，该方案可作为纯化AAV的通用捕获步骤。
该方法依赖于 AAV 血清型 2 （AAV2） 对硫酸乙酰肝素蛋白聚糖的内在亲和力。具体来说，该方案需要将编码所需 AAV 衣壳蛋白的质粒与 AAV2 的质粒共转染，从而产生结合了两种亲本血清型特性的镶嵌 AAV 载体。简而言之，在生产细胞裂解后，按照使用标准快速蛋白液相色谱 （FPLC） 系统的优化单步肝素亲和层析方案直接纯化含有 AAV 颗粒的混合物。纯化的AAV颗粒随后被浓缩，并在纯度和生物活性方面进行全面表征。该协议提供了一种简化且可扩展的方法，无需超速离心和梯度即可执行，从而产生干净且高病毒滴度。

Adeno-associated virus (AAV) vector is conquering its way as one of the most promising delivery systems in current gene therapy studies. Initially identified in 19651, AAV is a small, non-enveloped virus, with an icosahedral protein capsid of about 25 nm in diameter, harboring a single-stranded DNA genome. AAVs belong to the Parvoviridae family and to the Dependoparvovirus genus owing to their unique dependence on co-infection with a helper virus, such as herpes simplex virus or, more frequently, adenovirus, to complete their lytic cycle2,3.
The 4.7 kilobase genome of AAVs consists of two open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs) that form characteristic T-shaped hairpin ends4. ITRs are the only cis-acting elements critical for AAV packaging, replication, and integration, therefore being the only AAV sequences retained in recombinant AAV (rAAV) vectors. In this system, the genes necessary for vector production are supplied separately, in trans, allowing the gene of interest to be packaged inside the viral capsid5,6.
Each viral gene codifies different proteins through alternative splicing and start codons. Within the Rep ORF, four non-structural proteins (Rep40, Rep52, Rep68, and Rep78) are encoded, playing crucial roles in replication, site-specific integration, and encapsidation of viral DNA7,8. The Cap ORF serves as a template for the expression of three structural proteins differing from each other at their N-terminus, (VP1, VP2, and VP3), that assemble to form a 60-mer viral capsid at a ratio of 1:1:104,9. Additionally, an alternative ORF nested in the Cap gene with a nonconventional CUG start codon encodes an assembly-activating protein (AAP). This nuclear protein has been shown to interact with the newly synthesized capsid proteins VP1-3 and promote capsid assembly10,11.
Differences in the amino acid sequence of the capsid account for the 11 naturally occurring AAV serotypes and over 100 variants isolated from humans and non-human primate tissues7,12,13. Variations in the conformation of structurally variable regions govern the distinct antigenic properties and receptor-binding specificities of capsids from different strains. This results in distinct tissue tropisms and transduction efficiencies across different mammalian organs14.
Early production methods of rAAVs relied on adenovirus infection for helper purposes15,16,17,18,19. Despite being efficient and usually easy to produce on a large scale, several problems arise from this infection. Even after the purification and a heat-denaturing step for inactivation, adenoviral particles may still be present in AAV preparations, constituting an unwanted safety issue20. Moreover, the presence of denatured adenoviral proteins is unacceptable for clinical use. Other production strategies take advantage of recombinant herpes simplex virus strains engineered to bring the Rep/Cap and transgene into the target cells21 or of the baculovirus-insect cell system22. Although these systems offer advantages in terms of scalability and GMP compatibility, they still face similar problems.
The triple transfection method for rAAV production has been commonly adopted to easily overcome these issues. Briefly, the rAAV assembly relies on the transient transfection of cells with three plasmids encoding for: 1) the transgene expression cassette packed between the ITRs from the wild-type AAV2 genome (pITR); 2) the Rep/Cap sequences necessary for replication and virion assembly (pAAV-RC); and 3) the minimal adenoviral proteins (E1A, E1B, E4, and E2A) along with the adenovirus virus-associated RNAs required for the helper effect (pHelper)6,20,23. While plasmid transfection methods provide simplicity and flexibility for rAAV production in preclinical studies, these procedures have limitations in terms of scalability and reproducibility when applied to large-scale production. As an alternative approach, rAAV production can be achieved through the use of AAV producer cell lines (of both adherent and suspension growth), stably expressing either AAV Rep/Cap genes or Rep/Cap in combination with vector constructs. In these systems, the adenoviral helper genes are introduced through plasmid transfection. Even though this strategy improves the scalability of the cell culture process, it is technically complex and time-consuming21,24,25.
In either case, the producer cells are then lysed and subjected to one or multiple purification steps. Currently, the principal methods for purifying rAAVs include the use of cesium chloride (CsCl) or iodixanol ultra-high speed density gradient centrifugation followed, or not, by chromatography techniques26. The original purification scheme for viral precipitation used ammonium sulfate, followed by two or three rounds of ultracentrifugation through a CsCl gradient. The main advantages of this process include the possibility to purify all serotypes and the ability to physically separate full particles from empty capsids based on their different densities. This method, however, is elaborate, time-consuming, and has limited scalability, often resulting in poor yield and low sample quality27,28,29,30. Moreover, dialysis against a physiological buffer is often necessary before in vivo studies due to the toxic effects that CsCl can exert on mammals.
Iodixanol has also been used as an alternative iso-osmotic gradient medium to purify rAAV vectors, with advantages over CsCl from both safety and vector potency points of view. However, like CsCl, the iodixanol method presents some drawbacks related to the loading capacity of cell culture lysate (and thus the scalability of rAAV purification) and it remains a time-consuming and expensive method30,31.
To overcome these constraints, researchers turned their attention to chromatography techniques. In this regard, several purification approaches were developed that either incorporate affinity, hydrophobic, or ion-exchange chromatography methods. These methods rely on the biochemical properties of a particular serotype, including their natural receptors, or the charge characteristics of the viral particle32. For instance, the discovery that AAV2, AAV3, AAV6, and AAV13 preferably bind to heparan sulfate proteoglycans (HSPG), opened the possibility of using the closely related heparin in affinity chromatography purification. However, the binding sites to HSPG can differ among serotypes, mediating AAV attachment and infection of target cells in different ways2,33,34,35,36. On the other hand, AAV1, AAV5, and AAV6 bind to N-linked sialic acid (SA), while AAV4 uses O-linked SA2,14,34. Following the same rationale, a single-step affinity chromatography protocol for the purification of rAAV5 has also been developed based on the use of mucin, a mammalian protein highly enriched in SA37. Like heparin-based techniques, this purification is also dependent on the specific serotype being generated. Apart from heparin and mucin, other ligands have been explored for affinity chromatography, such as the A20 monoclonal antibody and camelid single-domain antibodies (AVB Sepharose and POROS CaptureSelect)22,23,38,39,40,41. Other innovative strategies to improve the previously existing purification methods involve the introduction of small modifications in the rAAV capsid to present specific binding epitopes. For instance, hexa-histidine-tagged or biotinylated rAAVs can be purified using ligands that target those epitopes (nickel nitrilotriacetic acid and avidin resins, respectively)42,43,44.
In an effort to broaden the desired characteristics of rAAVs, investigators have cross-dressed the virions by mixing their capsids. This is accomplished by supplying the capsid gene from two distinct AAV serotypes in equimolar or different ratios during production, giving rise to a capsid structure composed of a mixture of capsid subunits from different serotypes34,45,46,47,48,49,50. Previous studies provide physical evidence that co-expressing capsid proteins from AAV2 with AAV1 (1:1 ratio) and AAV2 with AAV9 (1:1 ratio) results in the generation of mosaic rAAV1/2 and rAAV2/9 vectors, respectively45,46,48. A major benefit of the generation of mosaic rAAVs is the capacity to integrate advantageous traits from different AAV serotypes, resulting in synergistic improvements in transgene expression and tropism, while maintaining other properties useful during rAAV production. Interestingly, certain mosaic variants even exhibit novel properties different from either parental virus46,47,49. By taking advantage of the heparin-binding ability of AAV2, mosaic rAAV vectors could potentially be generated and purified by mixing AAV2 with other natural or new AAV capsids generated by directed evolution and/or rational design. Nonetheless, previous studies have highlighted the importance of capsid subunit compatibility when attempting to assemble mosaic vectors. For instance, Rabinowitz and colleagues demonstrated that although the transcapsidation of AAV1, AAV2, and AAV3 led to an efficient co-assembly of mosaic capsids, the cross-dressing of these serotypes with AAV4 hindered the generation of stable virions34,45,47. Additionally, AAV1, AAV2, and AAV3 showed low compatibility with AAV5, given the reduced viral titers obtained when mixing these capsids at different ratios. Interestingly, mosaic rAAV2/5 showed decreased heparin-binding properties, while maintaining mucin-binding ability like parental AAV5. However, rAAV3/5 at a 3:1 ratio preserved the dual binding to heparin and mucin. Overall, the generation of new mosaic rAAVs with enhanced transduction, specific tropism, or low immunogenicity could greatly benefit from our understanding of capsid assembly and receptor interactions, with specific combinations still requiring thorough investigation and optimization.
In the present work, we describe a step-by-step protocol for the production and purification of rAAVs using an optimized heparin affinity chromatography method. rAAVs are produced by transient transfection and are purified using a fast protein liquid chromatography (FPLC) system. After the concentration of selected purified fractions, the resulting viral stocks are characterized in terms of titer, purity, intrinsic physical properties, and biological activity in vitro and in vivo. As a proof of concept, we demonstrate the improvements and applicability of this protocol for the generation of mosaic rAAV1/2 and rAAV2/9 vectors. The choice of each serotype was based on their strikingly different tropisms, potentially conferring their unique characteristics to the mosaic versions as well. AAV serotype 1, with an overall moderate tropism for the central nervous system (CNS), has the ability to transduce neurons and glia (to a lesser extent) and undergoes axonal transport in the anterograde and retrograde directions in vivo2,7,8. Additionally, AAV serotype 9 was chosen for its natural ability to cross the blood-brain barrier and target the CNS in both neonatal and adult mice51,52. Finally, AAV serotype 2 was selected given its ability to bind to heparin, allowing affinity chromatography33. The purified rAAV1/2 and rAAV2/9 particles combine the properties of both parental AAV serotypes and, therefore, constitute suitable vectors for the transduction of the CNS45,46,48,49.

作者聚焦：腺相关病毒载体生产和纯化的改进方法

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

In this work, we intend to develop an improved protocol for the production, purification, and characterization of mosaic adeno-associated viral vectors that could prove their worth in preclinical studies. Despite efforts to establish stable producer cell lines, transient transfection in mammalian cells is still the predominant workflow for AAV production. Then the AAVs are purified by ultra-high speed density gradient centrifugation, or by chromatography techniques that rely on the biochemical properties of viral particles.The commonly used methods to purify AAVs make use of cesium chloride, or iodixanol density gradient ultracentrifugation. Despite their advantages, they have some limitations. They are time-consuming, they have limited scalability, and they are often giving results to some vectors with low purity.Now, to overcome these constraints, several researchers have turned their attention to chromatography techniques. We describe a step-by-step protocol for the generation of mosaic rAAVs based on the heparin-binding ability of AAV2. This method renders highly pure and biologically active rAAVs ready to use in six days, presenting itself as a semi-automated, scalable, and cost-effective strategy to generate rAAVs for preclinical studies.Having demonstrated the applicability of this method for the generation of mosaic rAAVs, the production system can now be fine-tuned to achieve a better scalability and cost effectiveness by establishing a producer cell line. Additionally, we aim to remove empty particles by including a second polishing purification step.

Read this Scientific Article Protocol about Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors at JoVE.com

该手稿描述了使用优化的基于肝素的亲和色谱方法生成和纯化腺相关病毒载体的详细方案。它提供了一种简单、可扩展且具有成本效益的方法，无需超速离心。所得载体表现出高纯度和生物活性，证明了它们在临床前研究中的价值。

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

在这项工作中，我们打算开发一种改进的方案，用于花叶腺相关病毒载体的生产、纯化和表征，这可以证明它们在临床前研究中的价值。尽管努力建立稳定的生产细胞系，但哺乳动物细胞中的瞬时转染仍然是AAV生产的主要工作流程。然后通过超高速密度梯度离心或依赖于病毒颗粒的生化特性的色谱技术纯化AAV。纯化AAV的常用方法使用氯化铯或碘克沙醇密度梯度超速离心。尽管它们具有优点，但它们也有一些局限性。它们非常耗时，可扩展性有限，并且它们通常会对一些纯度低的载体产生结果。现在，为了克服这些限制，一些研究人员已将注意力转向色谱技术。我们描述了一种基于 AAV2 肝素结合能力生成镶嵌 rAAV 的分步方案。该方法可在六天内获得高纯度和生物活性的 rAAV，从而成为一种半自动化、可扩展且具有成本效益的策略，可生成用于临床前研究的 rAAV。在证明了该方法在生成镶嵌 rAAV 方面的适用性后，现在可以对生产系统进行微调，通过建立生产细胞系来实现更好的可扩展性和成本效益。此外，我们的目标是通过包括第二个抛光纯化步骤来去除空颗粒。

isolation-adeno-associated-viral-vectors-through-single-step-semi

Microsoft Bing

Research

JoVE Journal

Bioengineering

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

1.6K 次观看.  University of Coimbra. 在这项工作中，我们打算开发一种改进的方案，用于花叶腺相关病毒载体的生产、纯化和表征，这可以证明它们在临床前研究中的价值。尽管努力建立稳定的生产细胞系，但哺乳动物细胞中的瞬时转染仍然是AAV生产的主要工作流程。然后通过超高速密度梯度离心或依赖于病毒颗粒的生化特性的色谱技术纯化AAV。纯化AAV的常用方法使用氯化铯或碘克沙醇密度梯度超速离心?...