Erickson Sarah B, Pham Quan, Cao Xiaofu, Glicksman Jake, Kelemen Rachel E, Shahraeini Seyed S, Bodkin Sebastian, Kiyam Zainab, Chatterjee Abhishek
Department of Chemistry, Boston College, 2609 Beacon Street, 201 Merkert Chemistry Center, Chestnut Hill, Massachusetts 02467, United States.
Bioconjug Chem. 2024 Jan 17;35(1):64-71. doi: 10.1021/acs.bioconjchem.3c00411. Epub 2023 Dec 16.
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.
设计腺相关病毒（AAV）载体以实现对特定细胞类型的靶向转导，对于充分发挥其在人类基因治疗中的潜力至关重要。实现这一目标的一种有前景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）位点特异性掺入AAV衣壳蛋白中，为精确引入此类修饰提供了一种特别有吸引力的策略。在本研究中，我们表明，使用ncAA诱变，可以系统地改变AAV衣壳上重靶向配体（环RGD）的连接位点，以创建不同的缀合物结构，并且连接位点对重靶向效率有重大影响。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（每个衣壳的标记数）高度敏感，中等标记密度为cRGD介导的重靶向提供最佳活性。最后，我们开发了一种技术，通过以高保真度和效率将ncAA选择性掺入次要衣壳蛋白中，更精确地控制每个AAV衣壳的连接位点数量，从而可以合成具有不同化学计量的AAV缀合物。总之，该平台对衣壳修饰的位点和化学计量提供了无与伦比的控制，这将有助于开发具有理想特性的下一代AAV载体。
