**2. Emergence of rAAV as a therapeutic platform**

Adeno-associated virus was first discovered in 1965 as a contamination of rhesus monkey kidney cell cultures that were infected with adenovirus stocks [2]. Initially, the virus was called defective as it was incapable of self-replicating in the absence of a helper virus, adenovirus or herpesvirus. Later, it was classified as a member of the Parvovirus family, genus Dependovi‐ rus.

Further investigation determined that it is a small virus (approximately 20 nm) composed of an icosahedral protein capsid, which contains single-stranded DNA of 4.7 kb. The viral genome is flanked at each end by inverted terminal repeats sequences of 145 bp called ITRs. These sequences self-assemble into hairpin structures, generating a double-stranded sequence, which serves as a template for replication. The viral genome encodes for two proteins: Rep and Cap. Rep is required for single-stranded DNA replication and packaging. Cap is necessary to form the viral capsid and transduce cells efficiently.

AAV has never been associated with a disease or pathology [3]. Furthermore, due to the homology between the Rep-binding element present on the ITR, and the rAAVS1 sequence found on human chromosome 19, the viral genome can result in integration into the human genome [4]. This last feature is important because it shows that the virus can facilitate longterm expression of the viral genome. Additionally, specific integration of AAV in a defined locus minimizes the risks of mutagenesis due to random insertions, as other vectors do.

However, several genetic modifications of AAV have been performed in order to guarantee further safety for its translation into the clinic. First, the gene required for viral replication, called Rep, and the element required for site-specific integration were eliminated from the AAV genome. Therefore, this AAV variant, called recombinant vector (rAAV), will exist in an extrachromosomal state with very low integration efficiency into the genomic DNA, reducing the possibility of inducing random mutagenesis. Second, packaging of the rAAV genomic DNA was modified, incorporating a self-complementary rAAV genome rather than a singlestranded DNA genome [5]. Self-complementary virus differentiates from the recombinant virus in its ability to refold into double-stranded DNA, bypassing the synthesis of the second strand. This substitution has the advantage of reducing the lag time prior to transgene expression and consequently, increasing the biological efficiency of gene delivery. However, it significantly reduces the size of the transgene that could be inserted into the rAAV genome, from 5 kb to 3 kb. Third, several capsid serotypes that carry the rAAV genome have been identified and isolated.
