The insertion of sequences for functional peptides into the viral capsid is a well established and characterized possibility to retarget and modify the viral vectors. Over the last decade many different locations in the viral sequence have been evaluated for their quality to insert small peptides. We decided to use the well etablished 587 and the newly identifyed 453 integration site for our Virus Construction Kit.
For reasony of flexibility and usability we decided not to use single cutting restriction sites in stead of PCR-technology. This approach provids the advantage that all capsid coding constructs can be modifyed using ViralBricks in a single cloning step but also beared the risk to harm the functionality of the viral gene sequence. The functionality of the modified version with the introduced single cutting restriction sites was tested and the functionality was determined as comparable with the unmodifyed sequence.
In order to have these single cutting restriction sites two restriction sites had to be remooved from the Rep-protein coding region of pAAV-RepCap and the restriction sites had to be introduced into the Capsid coding region as show in the following figure.