Jang Seongmin, Shen Hao K, Ding Xiaozhe, Miles Timothy F, Gradinaru Viviana
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Mol Ther Methods Clin Dev. 2022 Jul 31;26:343-354. doi: 10.1016/j.omtm.2022.07.011. eCollection 2022 Sep 8.
Adeno-associated virus serotype 9 (AAV9) is a promising gene therapy vector for treating neurodegenerative diseases due to its ability to penetrate the blood-brain barrier. PHP.eB was engineered from AAV9 by insertion of a 7-amino acid peptide and point mutation of neighboring residues, thereby enhancing potency in the central nervous system. Here, we report a 2.24-Å resolution cryo-electron microscopy structure of PHP.eB, revealing conformational differences from other 7-mer insertion capsid variants. In PHP.eB, the 7-mer loop adopts a bent conformation, mediated by an interaction between engineered lysine and aspartate residues. Further, we identify PKD2 as the main AAV receptor (AAVR) domain recognizing both AAV9 and PHP.eB and find that the PHP.eB 7-mer partially destabilizes this interaction. Analysis of previously reported AAV structures together with our pull-down data demonstrate that the 7-mer topology determined by the lysine-aspartate interaction dictates AAVR binding strength. Our results suggest that PHP.eB's altered tropism may arise from both an additional interaction with LY6A and weakening of its AAVR interaction. Changing the insertion length, but not sequence, modifies PKD2 binding affinity, suggesting that a steric clash impedes AAVR binding. This research suggests improved library designs for future AAV selections to identify non-LY6A-dependent vectors and modulate AAVR interaction strength.
9型腺相关病毒（AAV9）因其穿透血脑屏障的能力，是一种用于治疗神经退行性疾病的很有前景的基因治疗载体。PHP.eB是由AAV9通过插入一个7氨基酸肽和邻近残基的点突变改造而来，从而增强了在中枢神经系统中的效力。在此，我们报告了PHP.eB分辨率为2.24埃的冷冻电子显微镜结构，揭示了其与其他7聚体插入衣壳变体的构象差异。在PHP.eB中，7聚体环呈弯曲构象，由工程化的赖氨酸和天冬氨酸残基之间的相互作用介导。此外，我们确定多囊蛋白2（PKD2）是识别AAV9和PHP.eB的主要腺相关病毒受体（AAVR）结构域，并发现PHP.eB的7聚体部分破坏了这种相互作用。对先前报道的腺相关病毒结构的分析以及我们的下拉数据表明，由赖氨酸-天冬氨酸相互作用决定的7聚体拓扑结构决定了AAVR的结合强度。我们的结果表明，PHP.eB改变的嗜性可能源于与LY6A的额外相互作用及其与AAVR相互作用的减弱。改变插入长度而非序列会改变PKD2的结合亲和力，这表明空间位阻冲突阻碍了AAVR的结合。这项研究为未来腺相关病毒的筛选提出了改进的文库设计，以鉴定非LY6A依赖性载体并调节AAVR相互作用强度。
