New tools at the intersection of genetic code expansion, virus engineering, and directed evolution
In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. We describe the application of this technology to the modification of adeno-associated virus (AAV) for the first time, enabling the generation of vectors with precisely re-engineered cell-targeting for gene therapy. Our UAA-AAV production platform enables the incorporation of UAAs bearing bio-orthogonal reactive handles into multiple specific sites on the virus capsid and their subsequent functionalization with various labeling molecules. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. Next, we used our unique UAA-AAV vector as a tool for the directed evolution of more active UAA incorporation machinery in mammalian cells. It is well known that the efficiency of unnatural amino acid mutagenesis in mammalian cells is limited by the suboptimal activity of the suppressor tRNAs currently in use. The ability to improve their performance through directed evolution can address this limitation, but no suitable selection system was previously available to achieve this. We have developed a novel platform for virus-assisted directed evolution of enhanced suppressor tRNAs (VADER) in live mammalian cells. Our system applies selective pressure for tRNA activity via the nonsense suppression-dependent production of UAA-AAV, and selectivity for the specific incorporation of interest comes from a novel virus purification strategy based on the unique chemistry of the UAA. We demonstrated > 10,000-fold selectivity for active tRNAs out of mock libraries and used this system to evolve libraries generated from the commonly used archaeal pyrrolysyl suppressor tRNA, ultimately identifying a variant which is three times as active as the original tRNA. Finally, we used next-generation sequencing to analyze the fate of every library member over the course of the selection and found that our VADER selection scheme is indeed selective for the enrichment of more active tRNA variants. This work provides a general blueprint for the evolution of better orthogonal suppressor tRNAs in mammalian cells.