Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

The overall goal of the following experiment is to create a highly diverse library of synthetic adeno associated viral capsids from which particles with a desired property can be selected. This is achieved by first fragmenting, cloned and selected a a v capsid genes via controlled DNAs one digestion to permit their subsequent reassembly into chimeric sequences via two PCR reactions. As a second step, the resulting PCR product is cloned back into a replication competent a a v construct to yield a plasmid library comprising at least 1 million different a a v capsid genes.

Next, this plasmid library is transfected into 2 9 3 T cells, together with an adenoviral helper plasmid in order to produce a chimeric virus library that can then be selected on target cells or in animals. The results show the successful molecular engineering and evolution of novel synthetic A A V vectors based on enhanced or more specific reporter gene expression in cultured cells or in adult mice. The main advantage of this technique over existing methods like peptide display is that DNA family shuffling has the potential to simultaneously alter and improve multiple features of the AV capsid from cell binding and intracellular trafficking to uncoating and gene expression in the nucleus.