AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery

Chemical approaches to probe and engineer AAV vectors

“…Fusion of larger proteins – such as antibody fragments and DARPINs – has also been used, but these are largely restricted to the N-terminus, or into the variable domain IV of the minor capsid protein through extensive linker optimization. 36–43 Such strategies have also been used to improve the selectivity of AAV vectors. However, fusion of a foreign protein to the N-terminus of the minor capsid protein VP2 disrupts the natural capsid architecture, since VP2 N-terminus is normally tucked inside the capsid.…”

A facile chemical strategy to synthesize precise AAV-protein conjugates for targeted gene delivery

“…We sought to compare the activity of our optimized AAV2-Nb chemical conjugate with such reported genetic fusion constructs between AAV2 and the same nanobody. [31][32][33][34][35][36][37] Following the strategies reported by the work of Koch-Nolte 31 and Buchholz et al, 34 we inserted the 5F7 nanobody sequence at the loop of the variable region IV of VP1, or fused it to the-N terminus of VP2 to generate constructs KO.VP1-Loop-Nb and KO.VP2-N-term-Nb, respectively (Figure 3i). SDS-PAGE analysis of the resulting virus preparations confirmed successful fusion/insertion of nanobody to the target minor capsid proteins (Figure 3j).…”

“…It has been possible to fuse small antibody-like proteins onto the N-terminus, or at an internal loop of minor capsid proteins of AAV. [31][32][33][34][35][36][37] However, only small and well-folded proteins are compatible with such fusion, and even then, it can disrupt capsid architecture and packaging efficiency. It has also been possible to attach proteins onto the AAV capsid using HUH tag and DNA linkers.…”