253. Expanded Packaging Capacity of AAV by Lumenal Charge Alteration

Abstract

Adeno-associated virus (AAV) is widely believed to be the safest viral vector for gene therapy. Moreover, the array of AAV serotypes available allows transduction of different tissues in vivo. However, one major limitation of AAV is the relatively small DNA packaging size (4,700 nt). Existing serotypes have been over-packaged with limited success and varying reproducibility. The consensus appears to be that AAV can be over packaged by ~10%, but with a concomitant reduction in both viral titers and in vivo transduction. We have taken a novel approach to increase the packaging limitations of AAV by focusing on the lumen of AAV where vector DNA interacts with the capsid. We have created a series of AAV capsid variants that alter the capsid lumenal charge using AAV-DJ as the scaffold. The variants add positively charged lysine and arginine residues at lumenally exposed sites within the capsid that, we believe, may allow for interaction and condensation of vector DNA increasing the packaging capacity and/or stabilizing the capsid in an over-packaged state. We present in vitro characterization of the library of capsid variants and demonstrate that variants with intermediate charge (+4 to +7 over wildtype) can be produced at high titer when over-packaged up to 6.2 kb. In addition, these variants can transduce HEK293 cells with a 5.6 kb length vector better than wildtype. Finally, these variants can completely encapsidate at least a 5.4 kb vector by alkaline Southern blot. To validat…