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Abstract：
Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (Tm) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in Tm, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.
摘要：
腺相关病毒（AAV）在单基因疾病的治疗中用作基因转移载体。一种变体，基于天然AAV2分离物的合理工程，指定的AAV-真实类型（AAV-TT），与野生型AAV2相比，在体内具有高度的神经嗜性，和基于它的向量，目前正在评估中枢神经系统的应用。AAV-TT与AAV2有14个氨基酸不同，包括R585S和R588T，先前显示的两个残基对于AAV2的硫酸乙酰肝素结合是必需的。AAV-TT和AAV2的衣壳结构通过低温电子显微镜在3.4和3.0µ分辨率下可视化，分别,突出了特定氨基酸差异的结构扰动。在不同pH条件下进行的差示扫描荧光法（DSF）表明，AAV2的解链温度（Tm）始终低于AAV-TT约5°C，但两者在pH5.5时显示出最大的稳定性，对应于晚期内体的pH，根据VP1u外化的要求提出，以促进内体逃逸。在AAV-TT的585和588位重新引入精氨酸导致Tm降低，证明在这些位置缺乏碱性氨基酸与衣壳稳定性有关。这些成果注解AAV2/AAV-TT残基的构造和热分歧,这解释了不同的细胞结合，转导，抗原反应性，以及两种病毒之间允许组织的转导。具体来说,这些数据表明,与AAV2相比,AAV-TT可能不利用聚糖受体介导的途径进入细胞,并且可能具有更低的抗原特性。
