“Sortagged” AAV: a novel chemo-enzymatic approach for site-specific ligand conjugation on AAV vectors

Adeno-associated virus (AAV) is a versatile viral vector that can be employed for gene delivery for the treatment of a variety of human diseases. Indeed, recombinant AAVs have demonstrated great advantages in pre-clinical and clinical applications, including in muscular, liver and central nervous system (CNS) disorders. Furthermore, AAV vectors have been extensively engineered at both the level of the transgene cassette, and at the capsid structural levels to improve the efficiency of gene delivery. However, depending on the capsid engineering technique used, there are still disadvantages concerning, for example, the ability of the modified vector to assemble correctly, or the fact that even when variants with a more desirable tropism are identified in vivo, the target receptor is usually unknown, which carries the risk of poor translatability and off-target effects. In this study, a chemogenetic protein ligation technology, based on sortase-mediated ligation, was developed in order to couple AAV vectors with a ligand of interest. Sortase-mediated ligation relies on the enzymatic activity of the bacterial enzyme sortase (SrtA) that cleaves a specific amino acid motif (LPXTG) in a target protein, and then ligates a nucleophile to it. AAV variants were obtained by introducing the sortase motif in two surface-accessible variable regions (VRs) of the AAV2 capsid, specifically VR-IV and VR-VIII. SrtA was then used to functionalize the “sortagged” AAV with a crosslinker-reactive moiety, in our case Amine-DBCO. This exposed moiety undergoes a click chemistry reaction with an azide-modified protein of interest, thus covalently ligating such protein to the AAV surface. As result, the AAV is decorated with the ligand of interest specifically at SrtA recognised sites. Different protein ligands can be chosen to either boost transduction efficiency or to redirect the viral vector to cells expressing the cognate receptor. In vitro optimization and in vivo validation confirm that chemogenetically-modified AAV2 vectors acquire new functional properties conferred by the conjugated ligand. This strategy is an innovative technique to engineer AAV capsid combining the accuracy of an enzymatic approach with the modularity of a click-chemistry reaction. The approach allows to conjugate virtually any ligand of choice to an AAV vector, and therefore open the possibility to develop new therapies for currently untreatable disease.