The ability to engineer adeno-associated virus (AAV) vectors for targeted transduction of specific cell types is critically important to fully harness their potential for human gene therapy. A promising approach to achieve this objective involves chemically attaching retargeting ligands onto the virus capsid. Site-specific incorporation of a bioorthogonal noncanonical amino acid (ncAA) into the AAV capsid proteins provides a particularly attractive strategy to introduce such modifications with exquisite precision. In this study, we show that using ncAA mutagenesis, it is possible to systematically alter the attachment site of a retargeting ligand (cyclic-RGD) on the AAV capsid to create diverse conjugate architectures and that the site of attachment heavily impacts the retargeting efficiency. We further demonstrate that the performance of these AAV conjugates is highly sensitive to the stoichiometry of capsid labeling (labels per capsid), with an intermediate labeling density providing optimal activity for cRGD-mediated retargeting. Finally, we developed a technology to more precisely control the number of attachment sites per AAV capsid by selectively incorporating an ncAA into the minor capsid proteins with high fidelity and efficiency, such that AAV conjugates with varying stoichiometry can be synthesized. Together, this platform provides unparalleled control over the site and stoichiometry of capsid modification, which will enable the development of next-generation AAV vectors tailored with desirable attributes.

中文翻译：

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精确操纵衣壳修饰的位点和化学计量能够优化功能性腺相关病毒缀合物

设计腺相关病毒（AAV）载体用于特定细胞类型的靶向转导的能力对于充分利用其用于人类基因治疗的潜力至关重要。实现这一目标的一种有前途的方法是将重靶向配体化学连接到病毒衣壳上。将生物正交非规范氨基酸 (ncAA) 定点掺入 AAV 衣壳蛋白中，提供了一种特别有吸引力的策略，可以精确地引入此类修饰。在这项研究中，我们表明，使用 ncAA 诱变，可以系统地改变 AAV 衣壳上重定向配体（环状 RGD）的附着位点，以创建不同的缀合物结构，并且附着位点严重影响重定向效率。我们进一步证明，这些 AAV 缀合物的性能对衣壳标记的化学计量（每个衣壳的标签）高度敏感，中间的标记密度为 cRGD 介导的重定向提供了最佳活性。最后，我们开发了一种技术，通过高保真度和效率选择性地将 ncAA 掺入次要衣壳蛋白中，更精确地控制每个 AAV 衣壳的附着位点数量，从而可以合成具有不同化学计量的 AAV 缀合物。总之，该平台提供了对衣壳修饰的位点和化学计量的无与伦比的控制，这将使开发具有所需属性的下一代 AAV 载体成为可能。