(310d) Construction of a Directed Evolution Library Platform for Modifying Adeno-Associated Viruses for Gene Therapy Applications

The utility of adeno-associated virus (AAV) as a gene transfer vector is being investigated clinically. As a result, AAV vector design has been an active area of research, with emphasis on modifying the capsid proteins to enhance specificity and/or efficiency of virus tropism. Directed evolution approaches to AAV capsid design are gaining momentum because of the difficulties of rational design approaches. In directed evolution, large pools (libraries) of AAV mutants are created from which desirable variants are selected. It is of particular interest to investigators to insert peptides, such as cell-surface receptor ligands, that can alter or enhance virus tropism, but the ideal location for insertion into the AAV capsid is often hard to predict a priori. Therefore, it is desirable to create libraries of peptides inserted into every possible location within the cap sequence and further useful if the crossover sites upstream and downstream of each insertion position contain additional sequence diversity. Transposon-based random insertion has been employed on the AAV capsid gene (cap), but limitations include a high frequency (94%) of frameshifted and/or misoriented inserts (non-viable mutants) as well as limited crossover diversity. Random domain insertion by DNAse I has been shown to yield libraries with diverse crossover sites, but still retains the same high frequency (94%) of non-viable mutants. In this work, we have developed a modular, filtered, platform library of AAV cap genes with peptides inserted in random positions throughout the gene. A plasmid carrying the cap ORF was randomly linearized with DNase I, blunted and ligated to a linker containing directional, unique, flanking restriction sites, encoding small amino acids. This created a library of plasmids with the linker randomly inserted throughout the cap ORF or vector backbone. Cap ORFs containing a single insert were isolated and mutants with frameshifted insertions and/or misoriented inserts were removed from the pool. Cap genes containing a single, in-frame, correctly oriented, randomly inserted linker were then subcloned into an AAV expression vector and the resultant library named pAAV_RaPID (Random Peptide Insertion by DNase). The pAAV_RaPID plasmid library can be used to quickly and easily subclone any peptide motif randomly into the AAV cap gene with a high frequency of viable mutants and broad crossover diversity to increase the probability of finding functional AAV variants in directed evolution studies. We have thus developed a user-friendly platform for quick and efficient construction of AAV directed evolution libraries for use in gene transfer vector development.