Fig. 2

Rational strategies of AAV capsid modification. Monoclonal antibody docking sites, capsid antigenic motifs (CAM) and lysine residues associated with ubiquitination, neddylation, SUMOylation or glycosylation in the capsid are mutated by site directed mutagenesis. Mutated capsid sequences are cloned in to a Rep-encoding plasmid to create a plasmid library that is then used to package a reporter encoding AAV genome. The AAV library is then put through several rounds of selection in vivo or in cell culture to enrich for AAV variants with desirable traits such as hepatocyte transduction and immune evasion. Chemical modification with compounds with hepatocyte affinity e.g. GalNAc, are attached to the AAV capsid to generate AAVs with enhanced hepatocyte transduction. Barcoded rational AAV vector evolution (BRAVE) involves bioinformatics identification of proteins with hepatocyte affinity. A DNA library encoding peptide fragments is generated and inserted in the specific positions of the capsid sequence within the Rep-encoding plasmid to produce a mutant capsid plasmid library. This is followed by packaging of the reporter encoding AAV genome bearing a unique nucleotide barcode sequence. Cre-recombination-based AAV targeted evolution (CREATE) involves insertion of random PCR generated fragments between specific capsid gene positions within the Rep-encoding plasmid to produce mutant capsid plasmid library. A reporter-encoding AAV genome with the poly A signal flanked by lox P sites is then packaged. The AAV library is selected in hepatocytes specific Cre expressing mice