scAAV3b-CAG-GFP

Human gene therapy, which began more than three decades ago, has now entered clinical use, with the first advanced therapeutics available on the European market and some clinical trials showing treatment outcomes comparable to or better than standard treatments. Vector systems based on nonpathogenic, replication-defective adeno-associated viruses (AAVs) have contributed to this success, particularly by mediating long-term transgene expression in postmitotic tissues such as liver, muscle, eye, and brain. AAV vectors transduce both dividing and terminally differentiated cells and lack intrinsic integrase activity, which reduces the risk of insertional mutagenesis. AAV vectors consist of a non-enveloped capsid that defines tissue preference and antigenic reactivity and a DNA vector genome that delivers a transgene expression cassette (TEC), i.e., the gene of interest including control elements. The vector genome contains inverted terminal repeats (ITRs) at both ends that represent the viral replication origin and packaging signal. It can be designed as single-stranded DNA, providing the sense and antisense versions of the TEC on separate molecules (single-stranded AAV vectors (ssAAV)), or it can be provided on a single molecule, separated by additional ITRs (self-complementary AAV vectors (scAAV)). For vector production, transient plasmid transfection protocols in mammalian cell lines are usually employed. While the so-called AAV vector plasmid provides the TEC flanked by the ITRs, thus serving as a template for the replication of the vector genome, the AAV helper plasmid introduces viral replication and packaging proteins (Rep proteins), viral capsid proteins (VP1, VP2, and VP3), and the assembly activation protein (AAP). The third "component" necessary for AAV vector production is the helper virus function, since AAV relies on the assistance of unrelated viruses (e.g., herpes viruses or members of the adenovirus (Ad) family) for replication and particle production.