## 1 Introduction  
Adeno-Associated Virus (AAV) is among the smallest viruses known, classified within the Parvovirus family and the genus of viruses dependent on helper viruses. Its discovery dates back to 1965 when it was found in adenovirus preparations, leading to its nomenclature [1]. AAV possesses a single-stranded DNA and lacks an envelope, exhibiting the simplest viral structure without autonomous replication capability. Its replication depends on a helper virus, typically adenovirus or herpes simplex virus (HSV), both of which have large double-stranded DNA genomes. Despite its prevalence, AAV has not been associated with autonomously causing diseases or pathological conditions in humans, as evidenced by epidemiological studies on human AAV infection. These studies indicate that a majority of adults carry antibodies to one or more AAV serotypes, yet no significant pathogenic impact has been identified [2; 3]. The AAV genome is characterized by a linear, single-stranded DNA molecule approximately 4.7 kb in length, encapsulated within a 20-25 nm icosahedral capsid. At each end of the genome are \(\sim\)145 base-pair Inverted Terminal Repeats (ITRs), which contain cis-acting elements essential for DNA replication initiation and viral particle packaging [4]. Between the ITRs lie two open reading frames: the Rep gene and the Cap gene, which encode the virus's essential proteins. In the genome, three virus-specific promoters, designated as p5, p19, and p40 based on their locations, play a crucial role in initiating the transcription of genetic information [5; 6]. These promoters facilitate the synthesis of mRNA that shares a common polyadennylation site (pA) (Figure 1A). The open reading frame (ORF)of Rep gene encodes several non-structural proteins essential for regulation, replication and transcription, including Rep78, Rep68, Rep52 and Rep40. Among them, Rep78 and Rep68 are essential for replication and integration, while Rep52 and Rep40 support genome packaging [7] (Figure 1B). The ORF of the Cap gene encodes three structural proteins: viral capsid protein 1 (VP1), VP2, and VP3. These assemble in a 1:1:10 ratio to form the AAV capsid, which protects the viral genome and facilitates entry into host cells [8] (Figure 1C). The AAV genome also encodes two accessory proteins associated with viral function and infection mechanisms: Assembly-Associated Protein (AAP), and Membrane-Associated Accessory Protein (MAAP) [9]. AAP encodes a capsid-associated protein that plays a crucial regulatory role in viral replication and assembly [9], while MAAP is involved in viral egress, genome packaging, and vector production [10; 11; 12] (Figure 1D). Notably, the AAV genome can integrate into a specific locus called AAVS1 on human chromosome 19, leading to latent infection. This integration is facilitated by the resemblance of ITR and Rep sequences between AAVS1 and AAV, contributing to the establishment of latent infections [13; 14]. Recombinant Adeno-Associated Virus (rAAV) is a viral vector crafted by substituting the Rep or Cap genes of the AAV genome with foreign genes and encapsidated in AAV capsids [15]. As a non-pathogenic virus, rAAV is able to express exogenous proteins safely, efficiently and persistently in vivo, properties that make it an ideal vector for the treatment of hereditary diseases [16; 17; 18], neurological disorders [19; 20] and certain cancers, highlighting its significant potential in gene therapy [21; 22; 23]. For example, patients with  
Figure 1: Schematic of AAV genome structure. (**A**) At both ends of the genome are two inverted terminal repeats (ITR), which are mainly used as the origin of virus replication and packaging signals. Three virus-specific promoters, named p5, p19 and p40 according to their positions, promote the synthesis of mRNA sharing a common polyadenylation site (pA). (**B**) The Rep gene encodes four kinds of protein, Rep78, Rep68, Rep52 and Rep40, which are required for virus replication and packaging. They are named after their molecular weights.