AAV1-CMV-Luc

In conventional rAAV, the rep and cap ORFs are replaced by a transgene expression cassette, leaving only the ITR sequences of the parental virus, which are the only cis-elements required for viral genome replication and its packaging into viral particles. The excised structural and nonstructural proteins can be provided in trans. Although rep- and cap-expressing packaging cell lines have been engineered, rAAV used in laboratories is typically produced by transient cell transfection using a vector plasmid and an AAV helper plasmid.

The vector plasmid contains the transgene expression cassette flanked by the viral ITRs. The two AAV-specific ORFs required in trans, rep and cap, are encoded on an AAV helper plasmid that does not carry the ITR sequences. These two plasmids are co-transfected with a third plasmid that provides the adenoviral functions required for AAV replication. Alternatively, rep, cap, and adenoviral helper genes can be combined on a single plasmid. In addition to providing the necessary helper viral genes by plasmid transfection, co-infection with wild-type adenovirus or herpes simplex virus can be performed, but this procedure produces AAV preparations contaminated with helper viruses. After transcription and translation of the Rep and VP proteins, the ITR-flanked transgene cassette of the vector plasmid is replicated and the single-stranded DNA molecule is encapsidated in preformed AAV capsids. The vector particles can then be purified from the cell lysate by density gradient centrifugation (CsCl or iodixanol) and/or column chromatography. The purified vector preparation also contains empty capsids, the amount of which depends on the packaging efficiency and the purification method.