Reducing Loss of AAV Inverted Terminal Repeats from Transfer Vector Plasmids Using the E. coli SSB Protein

Transient productions of recombinant AAV vectors in mammalian cells utilise a plasmid carrying a transgene flanked by AAV inverted terminal repeats (ITRs). ITRs are required for replication of the transfer vector, subsequent encapsidation and stability of the recombinant genome in transduced cells. During replication in E. coli, a proportion of these plasmids (~5-100%) suffer a partial deletion of an ITR, resulting in heterogeneous preparations; a universal problem in AAV vectorology. Recombinant AAV genomes flanked by ITRs containing deletions are inefficiently packaged into the capsid in mammalian cells, reducing target vector yields and giving unpredictable in vivo performance. These ITR deletions were postulated to be driven by formation of hairpin structures in the lagging strand of the DNA replication fork. In this model, the hairpin is bridged by an Okazaki fragment, resulting in daughter plasmids lacking part of the ITR sequence. We hypothesised that overexpression of the E. coli single stranded binding protein (SSB), that binds all single stranded DNA in the bacterium, would prevent hairpin formation. Cloning the ssb gene into an AAV transfer vector plasmid backbone so that SSB protein would be overexpressed in transformed bacteria resulted in greatly reduced ITR loss, potentially improving vector quality. Other factors may contribute, but addition of ssb to AAV transfer vector plasmids has largely solved the ITR instability issue, eased cloning steps for new AAV gene therapies and will potentially improve quality and yield of vector.