Precise Manipulation of the Site and Stoichiometry of Capsid Modification Enables Optimization of Functional Adeno-Associated Virus Conjugates.

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.

A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair.

We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNATrp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNATrp pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNATrp pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.

Labeling Proteins at Site-Specifically Incorporated 5-Hydroxytryptophan Residues Using a Chemoselective Rapid Azo-Coupling Reaction.

Chemoselective protein labeling is a valuable tool in the arsenal of modern chemical biology. The unnatural amino acid mutagenesis technology provides a powerful way to site-specifically introduce nonnatural chemical functionalities into recombinant proteins, which can be subsequently functionalized in a chemoselective manner. Even though several strategies currently exist to selectively label recombinant proteins in this manner, there is considerable interest for the development of additional chemoselective reactions that are fast, catalyst-free, use readily available reagents, and are compatible with existing conjugation chemistries. Here we describe a method to express recombinant proteins in E. coli site-specifically incorporating 5-hydroxytryptophan, followed by the chemoselective labeling of this residue using a chemoselective rapid azo-coupling reaction.

Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technology with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technology for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddition (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.

Synthesis at the interface of virology and genetic code expansion.

How a virus efficiently invades its host cell and masterfully engineers its properties provides valuable lessons and resources for the emerging discipline of synthetic biology, which seeks to create engineered biological systems with novel functions. Recently, the toolbox of synthetic biology has also been enriched by the genetic code expansion technology, which has provided access to a large assortment of unnatural amino acids with novel chemical functionalities that can be site-specifically incorporated into proteins in living cells. The synergistic interplay of these two disciplines holds much promise to advance their individual progress, while creating new paradigms for synthetic biology. In this review we seek to provide an account of the recent advances at the interface of these two research areas.

Production and Chemoselective Modification of Adeno-Associated Virus Site-Specifically Incorporating an Unnatural Amino Acid Residue into Its Capsid.

The ability to modify the capsid proteins of human viruses is desirable both for installing probes to study their structure and function, and to attach retargeting agents to engineer viral infectivity. However, the installation of such capsid modifications currently faces two major challenges: (1) The complex and delicate capsid proteins often do not tolerate large modifications, and (2) capsid proteins are composed of the 20 canonical amino acids, precluding site-specific chemical modification of the virus. Here, we describe a technology for generating adeno-associated virus (AAV) while incorporating an unnatural amino acid (UAA) into specific sites of the virus capsid. Incorporation of this UAA is generally tolerated well, presumably due to its small structural footprint. The resulting virus can be precisely functionalized at the site of UAA incorporation using chemoselective conjugation strategies targeted toward the azido side chain of this UAA. This technology provides a powerful way to modify AAV with unprecedented precision to both probe and engineer its entry process.

A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation.

Chemoselective modification of complex biomolecules has become a cornerstone of chemical biology. Despite the exciting developments of the past two decades, the demand for new chemoselective reactions with unique abilities, and those compatible with existing chemistries for concurrent multisite-directed labeling, remains high. Here we show that 5-hydroxyindoles exhibit remarkably high reactivity toward aromatic diazonium ions and this reaction can be used to chemoselectively label proteins. We have previously genetically encoded the noncanonical amino acid 5-hydroxytryptophan in both E. coli and eukaryotes, enabling efficient site-specific incorporation of 5-hydroxyindole into virtually any protein. The 5-hydroxytryptophan residue was shown to allow rapid, chemoselective protein modification using the azo-coupling reaction, and the utility of this bioconjugation strategy was further illustrated by generating a functional antibody-fluorophore conjugate. Although the resulting azo-linkage is otherwise stable, we show that it can be efficiently cleaved upon treatment with dithionite. Our work establishes a unique chemoselective "unclickable" bioconjugation strategy to site-specifically modify proteins expressed in both bacteria and eukaryotes.

Expanding the genetic code of mammalian cells.

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. Over the last few years, exciting progress has been made toward expanding the toolbox of genetically encoded UAAs in mammalian cells, improving the efficiency of their incorporation and developing innovative applications. Here, we provide our perspective on these recent developments and highlight the current challenges that must be overcome to realize the full potential of this technology.

Precise Photoremovable Perturbation of a Virus-Host Interaction.

Viruses utilize distinct binding interactions with a variety of host factors to gain entry into host cells. A chemical strategy is described to precisely perturb a specific molecular interaction between adeno-associated virus and its host cell, which can be rapidly reversed by light. This strategy enables pausing the virus entry process at a specific stage and then restart it rapidly with a non-invasive stimulus. The ability to synchronize the invading virus population at a discrete step in its entry pathway will be highly valuable for enabling facile experimental characterization of the molecular processes underlying this process. Additionally, adeno-associated virus has demonstrated outstanding potential for human gene therapy. This work further provides a potential approach to create therapeutic vectors that can be photoactivated in vivo with high spatial and temporal control.

A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus.

The ability to target the adeno-associated virus (AAV) to specific types of cells, by altering the cell-surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor-targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv ß3 integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site-dependent, underscoring the importance of achieving site-selective capsid modification. 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.