A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism
A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism
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DOI：
10.1073/pnas.1910061116
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发表时间：
2019-12-26
影响因子：
11.1
通讯作者：
Bjorklund, Tomas
中科院分区：
文献类型：
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作者：
Davidsson, Marcus;Wang, Gang;Bjorklund, Tomas
腺相关病毒(AAV)衣壳的修饰使重组载体的产生具有特定的性质和取向。到目前为止，大多数方法都依赖于随机筛选、丰富和偶然。这里探索的方法被称为BRAVER(条形码理性AAV载体进化)，使得只需在体内进行一轮筛选就可以有效地选择大规模的工程衣壳结构。这一方法与之前需要多代浓缩的方法形成了鲜明对比。通过这种大胆的方法，每个病毒颗粒在AAV衣壳表面显示一个源自蛋白质的已知功能的肽，并在包装的基因组中显示一个独特的分子条形码。来自体内单代筛选的RNA表达的条形码的测序允许同时映射来自数百个蛋白质的假定结合序列。使用勇敢的方法和基于隐马尔可夫模型的聚类，我们提出了25个具有精细性质的合成衣壳变体，例如特定类型神经元的逆行轴突运输，就像啮齿动物和人多巴胺能神经元所显示的那样。
Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.