Targeting AAV vectors to the central nervous system by engineering capsid-receptor interactions that enable crossing of the blood-brain barrier.
Targeting AAV vectors to the central nervous system by engineering capsid-receptor interactions that enable crossing of the blood-brain barrier.
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DOI：
10.1371/journal.pbio.3002112
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发表时间：
2023-07
期刊：
影响因子：
9.8
通讯作者：
中科院分区：
文献类型：
--
作者：
病毒已经进化出通过与多种细胞大分子相互作用结合并进入细胞的能力。我们设计了肽修饰的腺相关病毒（AAV）衣壳，其通过引入与在小鼠血脑屏障（BBB）上表达的2种蛋白质LY6A或LY6C1的从头相互作用而使脑萎缩。这些衣壳的体内向性是可预测的，因为它们依赖于其靶蛋白的细胞特异性和菌株特异性表达。这种方法在体外单轮筛选和体内二次验证中产生了数百种具有显著增强的中枢神经系统（CNS）向性的衣壳，从而与常规的多轮体内选择相比减少了动物的使用。通过该方法获得的可再现和定量数据使得能够对衣壳序列空间进行饱和诱变和机器学习（ML）引导的探索。值得注意的是，在我们的验证过程中，我们确定了几乎所有因其在小鼠中穿过BBB的能力而被选择的已发表的AAV衣壳都利用了LY6A或LY6C1蛋白，这在灵长类动物中不存在。这项工作表明，AAV衣壳可以直接靶向特定蛋白质，以产生具有已知作用机制和可预测向性的有效基因递送载体。利用重组病毒的基因治疗显示出治疗疾病的前景，但受到递送到疾病相关器官的效率低下的限制。这项研究表明，直接将腺相关病毒衣壳靶向血脑屏障上表达的特定蛋白质，可以快速产生具有可预测的体内向性的有效基因递送载体。
Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood–brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms. Gene therapy with recombinant viruses shows promise for treating diseases, but is constrained by the inefficiency of delivery to disease-relevant organs. This study shows that directly targeting adeno-associated virus capsids to specific proteins expressed on the blood-brain barrier can rapidly generate potent gene delivery vectors with predictable in vivo tropisms.
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