Name: isolation of next-generation gene therapy vectors through engineering, barcoding, and screening of adeno-associated virus (aav) capsid variants
Uploaded: 2022-10-18T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants at JoVE.com

D.G.非常感谢德国研究基金会（DFG）通过DFG合作研究中心SFB1129（Projektnummer 240245660）和TRR179（Projektnummer 272983813）以及德国感染研究中心（DZIF，BMBF;TTU-艾滋病毒 04.819）。

1. AAV2随机7-mer肽显示文库制备
注意：为了制备AAV2随机肽显示文库，将简并寡核苷酸合成为单链DNA，将其转换为双链DNA，酶解，连接受体质粒并电穿孔。
<ol><li>简并寡核苷酸的设计<ol><li>对简并寡核苷酸进行排序，避免密码子偏差。在寡核苷酸5'CAGTCGGCCAG AG W GGC （X01）7 GCCCAGGCGGCTGACGAG 3'中，X01对应于20个密码子，每个密码子编码20个氨基酸中的一个。W可以?...

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	<li>Wang, D., Tai, P. W. L., Gao, G. <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+vector+as+a+platform+for+gene+therapy+delivery.">Adeno-associated virus vector as a platform for gene therapy delivery.</a> Nature Reviews Drug Discovery. 18 (5), 358-378 (2019).</li><li>Muhuri, M., Levy, D. I., Schulz, M., McCarty, D., Gao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durability+of+transgene+expression+after+rAAV+gene+therapy.">Durability of transgene expression after rAAV gene therapy.</a> Molecular Therapy. 30 (4), 1364-1380 (2022).</li><li>Li, C., Samulski, R. 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在该协议中，概述了肽显示AAV衣壳工程和条形码AAV文库筛选以及文库组成和衣壳性能的生物信息学分析所需的步骤。该协议侧重于促进这些类型文库的生物信息学分析的步骤，因为大多数病毒学实验室在编程技能方面落后，以匹配他们在分子生物学技术方面的熟练程度。正如引言中所述，这两种类型的库在文献中都有广泛的描述，并且可以相对容易地复制。
作为第一步，概?...

基因转移疗法是在细胞中引入遗传物质以修复、替换或改变细胞遗传物质，以预防、治疗、治愈或改善疾病。体内和离体基因转移依赖于不同的递送系统，非病毒和病毒。病毒已经自然进化，可以有效地转导其靶细胞，并可用作递送载体。在基因治疗中使用的不同类型的病毒载体中，腺相关病毒已被越来越多地使用，因为它们缺乏致病性、安全性、低免疫原性，最重要的是它们能够维持长期、非整合的表达1，2，3。AAV基因治疗在过去十年中取得了相当大的成就;三种疗法已被欧洲药品管理局和美国食品和药物管理局批准用于人类3，4。一些临床试验也正在进行中，以治疗各种疾病，如血友病、肌肉、心脏和神经系统疾病，详见其他专题3。尽管取得了数十年的进步，但基因治疗领域近年来经历了一系列挫折4，最重要的是临床试验中的死亡5由于剂量限制性毒性而被搁置，特别是对于巨大的组织，如肌肉，或难以触及的组织，如大脑6。
目前用于临床试验的AAV载体属于天然血清型，但少数例外1。AAV工程为开发具有卓越器官或细胞特异性和效率的载体提供了机会。在过去的二十年中，已经成功应用了几种方法，例如肽显示，环交换，衣壳DNA洗牌，容易出错的PCR和靶向设计，以生成具有不同性质的单个AAV变体或其文库7。然后对它们进行多轮定向进化，以选择其中具有所需特性的变体，如其他地方1，3所述。在所有衣壳进化策略中，肽显示AAV文库使用最广泛，这是由于一些独特的性质：它们相对容易生成，并且可以实现高多样性和高通量测序，从而允许跟踪它们的进化。
第一个成功的肽插入AAV文库是在大约20年前描述的。在第一个中，Perabo等人8构建 了一个修饰的AAV2衣壳文库，其中随机生成的寡核苷酸池入质粒中，位置对应于VP1衣壳蛋白的氨基酸587，在从衣壳突出的三倍轴上。使用腺病毒混合感染，AAV文库通过多轮选择进化，最终的重新靶向变体被证明能够转导亲本AAV28难治的细胞系。此后不久，Müller等人9引入了 用于文库生产的两步系统，这是对该协议的重大改进。最初，质粒文库与腺病毒辅助质粒一起用于产生含有嵌合衣壳的AAV文库。该AAV穿梭文库用于感染低感染多重性（MOI）的细胞，目的是为每个细胞引入一个病毒基因组。与腺病毒的混合感染可确保产生具有匹配基因组和衣壳9的AAV。大约十年后，Dalkara10 使用 体内 定向进化创造了7m8变体。该变体具有10个氨基酸插入（LALGETTRPA），其中三个充当接头，并在玻璃体内注射后有效地靶向视网膜外层10。这种工程衣壳是一个非凡的成功故事，因为它是迄今为止为数不多的进入临床的工程衣壳之一11。
随着二代测序（NGS）技术的引入，该领域经历了第二次推动。Adachi et al.12 在 2014 年和 Marsic et al.13 在 2015 年的两篇出版物展示了 NGS 以高精度跟踪条形码 AAV 衣壳文库分布的能力。几年后，条形码区域的NGS适应肽插入区域以跟随衣壳的演变。Körbelin等人14进行了NGS引导的筛选，以确定基于肺靶向AAV2的衣壳。NGS分析有助于计算三个评分：选择轮之间的富集评分，确定组织特异性的一般特异性评分，最后是综合评分14。Gradinaru实验室15在同一年发表了基于Cre重组的AAV靶向进化（CREATE）系统，该系统有助于细胞类型特异性选择。在这个系统中，衣壳文库携带一个Cre可逆开关，因为polyA信号两侧有两个loxP位点。然后将AAV文库注射到Cre小鼠中，其中polyA信号仅在Cre+细胞中倒置，为反向PCR引物与衣壳基因内的正向引物结合提供模板。这种高度特异性的PCR救援能够鉴定AAV-PHP。可以穿过血脑屏障的B变体15。该系统进一步发展为M-CREATE（Multiplexed-CREATE），其中NGS和合成文库生成被集成到管道16中。
来自Maguire实验室17的基于RNA的改进版本iTransduce允许在DNA水平上选择功能转导细胞并表达其基因组的衣壳。肽显示文库的病毒基因组包括由普遍存在的启动子控制的Cre基因和在p41启动子控制下的衣壳基因。将该文库注射到tdTomato上游具有loxP-STOP-loxP盒的小鼠中。用AAV变体转导的细胞，这些变异体表达病毒基因组，因此Cre表达tdTomato，并与细胞标记结合，可以进行分类和选择17。类似地，Nonnenmacher等人18 和Tabebordbar等人19 将衣壳基因库置于组织特异性启动子的控制之下。注射不同动物模型后，使用病毒RNA分离衣壳变体。
另一种方法是使用条形码来标记衣壳库。Björklund实验室20 使用这种方法对肽插入衣壳文库进行条形码编码，并开发了条形码理性AAV载体进化（BRAVE）。在一个质粒中，将Rep2Cap盒克隆在倒置末端重复序列（ITR）侧向表达黄色荧光蛋白（YFP）的条形码标记转基因旁边。使用 帽 末端和条形码开头之间的loxP位点， 体外 Cre重组产生足够小的NGS片段，从而允许肽插入与唯一条形码（查找表，LUT）相关联。使用质粒文库进行AAV生产，并在 体内 应用后再次使用NGS20筛选mRNA中表达的条形码。当衣壳文库包含整个衣壳基因的变体（即洗牌文库）时，需要使用长读长测序。一些小组使用条形码来标记这些不同的库，这使得NGS具有更高的读取深度。Kay 实验室21 用 帽 polyA 信号下游的条形码标记了高度多样化的衣壳洗牌文库。第一步，生成条形码质粒文库，并将洗牌衣壳基因文库克隆到其中。然后，MiSeq（短读长，高读深）和PacBio（长读长，低读深）NGS以及Sanger测序的组合用于生成他们的LUT21。2019年，Ogden和来自ChurchLab 22 的同事使用在每个位置都有单点突变、插入和缺失的文库描绘了AAV2衣壳对多种功能的适应性，最终实现了机器引导设计。为了生成文库，合成衣壳基因的较小片段，用条形码标记，进行下一代测序，然后克隆到完整的衣壳基因中。NGS数据用于生成LUT。然后仅使用条形码和短读取序列对库进行筛选，这反过来又允许更高的读取深度22。
条形码文库主要用于筛选已知、天然和工程变体的池，经过几轮衣壳文库的选择或独立于衣壳进化研究。这种文库的优点是有机会筛选多个衣壳，同时减少动物数量并最大限度地减少动物之间的变异。将这项技术引入AAV领域的第一批研究是在大约十年前发表的。Nakai实验室12用一对12 核苷酸条形码标记了191个双丙氨酸突变体，覆盖AAV9的VP1上的氨基酸356至736。使用NGS，在 体内筛选 文库的半乳糖结合和其他性质12。Marsic及其同事在1年后使用双条带分析描绘了AAV变体的生物分布13。最近一项针对非人灵长类动物的研究比较了使用不同递送途径的29个衣壳在中枢神经系统中的生物分布23。我们的实验室最近发布了183种变体的条形码AAV库屏幕，其中包括天然和工程AAV。DNA和RNA水平上的这些筛选导致在小鼠中鉴定出高度肌致性AAV变体24以及在小鼠大脑中显示出高细胞类型特异性的其他变体25。
在这里，我们描述了这项工作中使用的方法，并对其进行了扩展，以包括AAV肽显示文库的筛选。这包括生成AAV2肽显示文库，用于定量的数字液滴PCR（dd-PCR）方法，最后是用于分析AAV变体的NGS管道，部分基于Weinmann及其同事的工作24。最后，提供了条形码AAV文库的生成和同一出版物中使用的NGS管道的描述。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplification primer</td>
			<td>ELLA Biotech (Munich, Germany)</td>
			<td>-</td>
			<td>Second-strand synthesis of oligonucleotide insert</td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 Reagents</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>5067-1504</td>
			<td>DNA fragment validation</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>G2938C</td>
			<td>DNA fragment validation</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA Mini Kit&#160;</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>80204</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 Reagents</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>5067-1504</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies (Santa Clara, CA, USA)</td>
			<td>G2938C</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>BC-seq fw:&#160;</td>
			<td>IDT (San Joce, CA, CA, USA)</td>
			<td>ATCACTCTCGGCATGGACGAGC&#160;</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>BC-seq rv:&#160;&#160;</td>
			<td>IDT (San Joce, CA, CA, USA)</td>
			<td>GGCTGGCAACTAGAAGGCACA&#160;</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Millipore Sigma (Burlington, MA, USA)</td>
			<td>44-420-3250ML</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>BglI</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>R0143</td>
			<td>Digestion of double-stranded insert</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1851196</td>
			<td>dd-PCR cycler</td>
		</tr>
		<tr>
			<td>dNTPS&#160;</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>N0447S</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for probes (no dUTP)</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863024</td>
			<td>dd-PCR supermix</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863005</td>
			<td>dd-PCR droplet generation oil</td>
		</tr>
		<tr>
			<td>DG8 Cartridges for QX100 / QX200 Droplet Generator</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1864008</td>
			<td>dd-PCR droplet generation cartridge</td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863051</td>
			<td>dd-PCR cartridge holder</td>
		</tr>
		<tr>
			<td>Droplet Generator DG8 Gasket</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1863009</td>
			<td>dd-PCR cover for cartridge</td>
		</tr>
		<tr>
			<td>ddPCR Plates 96-Well, Semi-Skirted</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>12001925</td>
			<td>dd-PCR 96-well plate</td>
		</tr>
		<tr>
			<td>E.cloni 10G SUPREME Electrocompetent Cells</td>
			<td>Lucigen (Middleton, WI, USA)</td>
			<td>60081-1</td>
			<td>Electrocompetent cells</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes, 1mm</td>
			<td>Biozym Scientific (Oldendorf, Germany)</td>
			<td>748050</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>GAPDH primer/probe mix</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>Mm00186825_cn</td>
			<td>Taqman qPCR primer</td>
		</tr>
		<tr>
			<td>Genepulser Xcell</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1652660</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems (Waltham, MA, USA)</td>
			<td>4368814</td>
			<td>cDNA reverse transcription</td>
		</tr>
		<tr>
			<td>ITR_fw</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>GGAACCCCTAGTGATGGAGTT (https://signagen.com/blog/2019/10/25/qpcr-primer-and-probe-sequences-for-raav-titration/)</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>ITR_rv</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>CGGCCTCAGTGAGCGA (https://signagen.com/blog/2019/10/25/qpcr-primer-and-probe-sequences-for-raav-titration/)</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>ITR_probe</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>HEX-CACTCCCTCTCTGCGCGCTCG-BHQ1 (https://signagen.com/blog/2019/10/25/qpcr-primer-and-probe-sequences-for-raav-titration/)</td>
			<td>dd-PCR probe</td>
		</tr>
		<tr>
			<td>Illumina NextSeq 500 system</td>
			<td>Illumina Inc (San Diego, CA, USA)</td>
			<td>SY-415-1001</td>
			<td>NGS Library sequencing</td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix (2X)*</td>
			<td>Roche AG (Basel, Switzerland)</td>
			<td>KK2600 07958919001</td>
			<td>NGS sample prepration</td>
		</tr>
		<tr>
			<td>MagnaBot 96 Magnetic Separation Device</td>
			<td>Promega GmbH (Madison, WI, USA)</td>
			<td>V8151</td>
			<td>Sample prepration for NGS library</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 spectrophotometer</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>ND-2000</td>
			<td>Digestion of double-stranded insert</td>
		</tr>
		<tr>
			<td>NGS_frw</td>
			<td>Sigma-Aldrich (Burlinght, MA, USA)</td>
			<td>GTT CTG TAT CTA CCA ACC TC</td>
			<td>NGS primer</td>
		</tr>
		<tr>
			<td>NGS_rev</td>
			<td>Sigma-Aldrich (Burlinght, MA, USA)</td>
			<td>CGC CTT GTG TGT TGA CAT C</td>
			<td>NGS primer</td>
		</tr>
		<tr>
			<td>NextSeq 500/550 High Output Kit (75 cycles)</td>
			<td>Illumina Inc (San Diego, CA, USA)</td>
			<td>FC-404-2005</td>
			<td>NGS Library sequencing</td>
		</tr>
		<tr>
			<td>Ovation Library System for Low Complexity Samples Kit&#160;</td>
			<td>NuGEN Technologies, Inc. (San Carlos, CA, USA)</td>
			<td>9092-256</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>PX1 Plate Sealer&#160;</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1814000</td>
			<td>dd-PCR plate sealer</td>
		</tr>
		<tr>
			<td>Pierceable Foil Heat Seal</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1814040</td>
			<td>dd-PCR sealing foil</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA-Polymerase</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>F530S</td>
			<td>Second-strand synthesis of oligonucleotide insert</td>
		</tr>
		<tr>
			<td>PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)</td>
			<td>Polysciences, Inc. (Warrington, PA, USA)</td>
			<td>24765-1G</td>
			<td>AAV library preparation</td>
		</tr>
		<tr>
			<td>ProNex Size-Selective Purification System</td>
			<td>Promega GmbH (Madison, WI, USA)</td>
			<td>NG2002</td>
			<td>Sample prepration for NGS library</td>
		</tr>
		<tr>
			<td>Phusion Hot Start II Polymerase&#160;</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>F549L</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche AG (Basel, Switzerland)</td>
			<td>5963117103</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>pRep2Cap2_PIS</td>
			<td></td>
			<td></td>
			<td>ITR-Rep2Cap2-ITR vector. Peptide insertion site within the Cap2 ORF, manufactured/prepared in the lab&#160;</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1864002</td>
			<td>dd-PCR droplet generator</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio-Rad (Hercules, CA, USA)</td>
			<td>1864003</td>
			<td>dd-PCR droplet analysis</td>
		</tr>
		<tr>
			<td>QIAquick Nucleotide Removal Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>28306</td>
			<td>Second-strand synthesis of oligonucleotide insert purification</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>28704</td>
			<td>Plasmid vector purification</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>12162</td>
			<td>Plasmid library DNA preparation</td>
		</tr>
		<tr>
			<td>Qiaquick PCR Purification kit</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>28104</td>
			<td>Sample prepration for NGS library</td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>Invitrogen (Waltham, MA, USA)</td>
			<td>Q32857</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS</td>
			<td>Thermo Fischer Scientific (Waltham, MA, USA)</td>
			<td>Q32851</td>
			<td>NGS Library preparation</td>
		</tr>
		<tr>
			<td>QuantiFast PCR Master Mix</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>1044234</td>
			<td>Taqman qPCR</td>
		</tr>
		<tr>
			<td>rep_fw</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>AAGTCCTCGGCCCAGATAGAC</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>rep_rv</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>CAATCACGGCGCACATGT</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>rep_probe</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>FAM-TGATCGTCACCTCCAACA-BHQ1</td>
			<td>dd-PCR probe</td>
		</tr>
		<tr>
			<td>RNase-free DNase</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>79254</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>SfiI</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>R0123</td>
			<td>Digestion of vector</td>
		</tr>
		<tr>
			<td>5 mm, steel Beads</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>69989</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>TRIMER-oligonucleotides</td>
			<td>ELLA Biotech (Munich, Germany)</td>
			<td>-</td>
			<td>Degenerate oligonucleotide</td>
		</tr>
		<tr>
			<td>T4 Ligase</td>
			<td>New England Biolabs (Ipswich, MA, USA)</td>
			<td>M0202L</td>
			<td>Plasmid library ligation</td>
		</tr>
		<tr>
			<td>TissueLyserLT</td>
			<td>Qiagen (Venlo, Netherlands)</td>
			<td>85600</td>
			<td>DNA/RNA extraction</td>
		</tr>
		<tr>
			<td>YFP_fw</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>GAGCGCACCATCTTCTTCAAG</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>YFP_rv</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>TGTCGCCCTCGAACTTCAC</td>
			<td>dd-PCR primer</td>
		</tr>
		<tr>
			<td>YFP_probe</td>
			<td>IDT (San Joce, CA, USA)</td>
			<td>FAM-ACGACGGCAACTACA-BHQ1</td>
			<td>dd-PCR probe</td>
		</tr>
		<tr>
			<td>Zymo DNA Clean &#38; Concentrator-5 (Capped)</td>
			<td>Zymo research (Irvine, CA, USA)</td>
			<td>D4013</td>
			<td>Vector and Ligation purification</td>
		</tr>
	</tbody>
</table>

isolation of next-generation gene therapy vectors through engineering, barcoding, and screening of adeno-associated virus (aav) capsid variants

源自腺相关病毒（AAV）的基因递送载体是治疗遗传疾病的最有前途的工具之一，令人鼓舞的临床数据和几种AAV基因疗法的批准证明了这一点。AAV载体成功的两个主要原因是（i）先前分离出具有不同特性的各种天然存在的病毒血清型，以及（ii）随后建立了强大的分子工程技术，并在高通量中再利用。最近实施的策略进一步提高了这些技术的潜力，用于在DNA和RNA水平上对选定的AAV衣壳进行条形码编码，允许它们在单个动物的所有主要器官和细胞类型中进行全面和平行的 体内 分层。在这里，我们提出了一个包含这组互补途径的基本管道，使用 AAV 肽显示来代表可用衣壳工程技术的各种库。因此，我们首先描述了生成AAV肽显示库的关键步骤，用于 体内 选择具有所需特性的候选药物，然后演示如何对最有趣的衣壳变体进行条形码编码以进行二次 体内 筛选。接下来，我们举例说明了创建用于下一代测序（NGS）文库的方法，包括条形码扩增和衔接连接，最后概述了NGS数据分析中最关键的步骤。由于这里报告的协议是通用和适应性强的，研究人员可以很容易地利用它们来丰富他们最喜欢的疾病模型中的最佳AAV衣壳变体和基因治疗应用。

生成 AAV2 肽显示库。 作为选择工程AAV的第一步，描述了质粒文库的生成。肽插入片段是使用简并引物产生的。将密码子的组合从64个减少到20个具有消除终止密码子和促进NGS分析的优点，方法是减少DNA上的文库多样性，而不是蛋白质水平上的文库多样性。寡核苷酸插入片段以单链DNA形式购买（图1），通过PCR反应将其转化为双链DNA。该反应的质量在生物分析仪中?...

Watch this Scientific Journal Video about Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants at JoVE.com

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants

AAV肽显示文库生成，随后通过条形码对具有新颖特性的候选物进行验证，以创建下一代AAV。

通过工程、条形码和筛选腺相关病毒 （AAV） 衣壳变体分离下一代基因治疗载体

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by ...

该协议通过实现病毒的工程化、多样化和分层，缩小了病毒的自然进化与其用作人类基因治疗重组载体之间的显着差距。该技术允许对新型、分离或工程化的腺相关病毒（简称AAV）变体进行高通量并行筛选，从而节省动物数量、工作、成本和时间。已鉴定的AVV衣壳变体可以提高基于AAV的治疗性转基因递送的功效和特异性，从而减少所需的病毒剂量。这可以提高基因治疗的安全性和适用性。同样的方法也可以用于其他基因递送载体的衣壳或启动子工程，从而提高我们对它们的生物学及其在基因治疗中的应用的理解。帮助演示该程序的将是博士生Jonas Becker和Jixin Liu，博士后Joanna Szumska和Margarita Zayas，以及我实验室的研究助理Emma Gerstmann和Ellen Widtke。首先使用 Python 3 和 Biopython 分析 NGS 测序数据。NGS分析由两个步骤组成。第一步，使用脚本 1 在序列文件中搜索满足特定条件（如侧翼序列、长度和位置）的序列，以及提供所需信息的配置文件。对于第二步，使用第二个脚本以及配置和 zuordnung 从 AGWGGC 序列开始翻译提取的序列。提供所需信息的 TXT 文件。准备两个文件夹：脚本和数据。将排序产生的 Gzip 压缩文件复制到数据文件夹。然后将 Python 和配置文件复制到脚本文件夹中，如文本手稿中所述。在运行脚本之前，请打开 zuordnung。txt 文件并添加两个制表符分隔的列。在第一列中输入 Gzip 文件的名称，在第二列中输入所需的最终名称。按照文本手稿中的说明更改配置文件中的变量。使用特定命令，启动变异序列检测和提取。输出将是TXT文件，其中包含提取的DNA序列及其读取次数。此文件的标头包含统计数据，这些数据将传输到以下文件。这些 TXT 数据将成为第二个脚本的输入文件，其中 DNA 序列被翻译、排名和分析。使用特定命令，按照文本手稿中所述，启动脚本一的文本输出文件的PV翻译和分析。第二个脚本的输出文件将使用 zuordnung 中查找表的第二列命名。TXT 带有基于分析类型的扩展名。确保三个输出文件在第一行和第一列中包含统计数据，其中包含输入文本文件中每个 DNA 序列的索引。其余列应由DNA序列，读取次数，正向或反向读取以及翻译的肽序列组成。无效序列应具有 NA，并且在最后两列中无效。根据用户的需求，使用可用的软件可视化输出文件。为了使用 Python 3 中的自定义代码对 NGS 测序数据进行分析，工作流程包括检测由侧翼序列、其长度和位置引导的条形码序列，以及分析一组组织的条形码富集和分布。准备两个文件夹：脚本和数据。将排序产生的 Gzip 压缩文件复制到数据文件夹。然后将 python 和配置文件复制到脚本文件夹中，如文本手稿中所述。在执行脚本之前，请创建两个制表符分隔的文本文件：衣壳方差。txt 文件，其中包含分配给 AAV 衣壳变体名称的条形码序列以及污染。TXT 文件，其中包含可能来自污染的条形码序列。最后，编辑配置文件以包括包含排序数据的文件夹路径信息、条形码侧翼区域的序列、它们的位置以及条形码检测的窗口大小。使用相应的命令使用提供的路径和配置文件执行条形码检测脚本。此命令执行的输出将是 TXT 文件，其中包含每个衣壳变体的重新计数以及从原始数据中恢复的读取总数。评估条形码AAV衣壳在组织或器官中的分布，在左旋律中。TXT文件中，将从条形码检测运行中获得的每个文本文件的名称分配给组织或器官名称。在第一列中添加文本文件的名称，并在制表符分隔的分配中添加相应的组织或器官名称。创建一个器官。TXT文件，其中包含目标上和目标外器官的名称列表，这些名称对应于分配中给出的名称。txt 文件。然后创建normalization_organ。TXT 和normalization_variant。TXT 选项卡分隔的文本文件，其中包含所有衣壳变体以及所有器官和组织的规范化值。在带有normalization_organ的第一列中。txt 文件，写下为每个器官给出的名称，第二列带有相应组织的归一化值。填充normalization_variant的第一列。TXT 文件包含衣壳名称列表，第二列包含池库中每个衣壳的读取计数的规范化值。通过指定所有其他文件的完整路径来编辑配置文件，并使用此特定命令执行条形码分析脚本。条形码分析脚本输出多个文件，例如基于前面描述的多个归一化步骤在不同组织内衣壳分布的相对浓度或RC值的文本文件，以及将文本文件数据合并为合并矩阵数据的电子表格文件。可视化数据并对矩阵数据进行聚类分析，如文本手稿中所述。脚本将输入相对浓度。XLS 文件并生成分层聚类热图和主成分分析的两个图。若要修改图或 PNG 参数，请打开 R 脚本并按照注释部分中的说明进行操作。AAV2 rep基因的定量区域显示99.2%的ITR呈阳性，表明AAV衣壳含有整个病毒基因组。在所有三组中，基于特定于库的特征序列提取的读段约占总读段的94%，表明质量良好。其中，超过99%是有效的PV读取，超过99%的有效PV读取是唯一的，这表明该库具有高内部多样性的平衡。热图层次结构的两个主要分支反映了衣壳变体转导效率的差异。具有大多数衣壳变体的左分支包括所有衣壳，这些衣壳在大多数组织中显示出较高的相对浓度值。除了惊人的高肝脏特异性外，其他三种衣壳在横膈膜、骨骼肌、二头肌和大脑中表现出特异性。分层聚类的右分支包括衣壳变体，其整体转导效率较低，在十二指肠和胰腺中最为明显。原始亚群的主成分分析形成了具有高肝脏特异性的衣壳变异簇，并概述了VAR60衣壳出色的肌肉嗜性。尝试此协议时，您需要注意键入错误。与 Linux 相比，如果您使用的是 Windows 命令提示符，语法也会有所不同。在鉴定出新的AAV变体后，需要在已故或更大的动物模型中验证治疗和转化潜力。该技术能够在相同条件下直接并排比较AAV变体，并且用途广泛，为其在大型动物甚至人类中的翻译铺平了道路。

Research

JoVE Journal

Bioengineering

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

科研

生物工程

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

新型原子力显微镜基于生物淘选的形态特殊试剂对TDP-43变型肌萎缩侧索硬化症隔离

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

机器人镜治疗系统偏瘫武器功能恢复

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

小鼠脂肪组织来源的微血管片段作为血管化单元的分离组织工程

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

通过从光响应聚合物纳米载体的siRNA释放的时空控制预测基因沉默

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Studying Cavitation Enhanced Therapy

研究屈化强化疗法

Interest in the therapeutic applications of ultrasound is significant and growing, with potential clinical targets ranging from cancer to ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

通过工程、条形码和筛选腺相关病毒 （AAV） 衣壳变体分离下一代基因治疗载体

通过工程、条形码和筛选腺相关病毒（AAV）衣壳变体分离下一代基因治疗载体