posted on 2023-12-16, 17:03authored bySarah
B. Erickson, Quan Pham, Xiaofu Cao, Jake Glicksman, Rachel E. Kelemen, Seyed S. Shahraeini, Sebastian Bodkin, Zainab Kiyam, Abhishek Chatterjee
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.