**2. Adeno-associated virus (AAV)**

Gene therapy protocols using recombinant viral vectors have proven potentially useful in molecular medicine. AAV is one of the most actively investigated gene therapy vehicles. It is a small (25 nm), non-enveloped virus composed by an icosahedral capsid that contains a single-stranded, 4.7-kb DNA genome. AAV genome is comprised of two genes rep and cap that are flanked by two palindromic inverted terminal repeats (ITR). Rep encodes for proteins associated with replication of the viral DNA, packaging of AAV genomes, and viral genome integration in the host DNA [15]. Cap encodes for the three proteins that form the capsid. In recombinant AAV vectors (rAAV), DNA sequences of interest between the AAV inverted terminal repeats (ITRs) are cloned, eliminating the entire coding sequence of the wt AAV genome. In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells [16]. During AAV assembly, rep and cap genes are provided in trans together with the adenoviral helper proteins required for AAV genome replication and packaging [17, 18]. The most common method of rAAV production is by triple transfection of HEK293 cells with three plasmids: one containing the transgene expression cassette flanked by the viral ITRs, a second packaging plasmid expressing the rep and cap genes and a third plasmid encoding for adenoviral helper genes [17, 19].

To date, 13 different AAV serotypes and 108 isolates have been identified and classified [15, 20]. AAV2 was one of the first AAV serotypes identified and characterized, including the sequence of its genome. As a result of the detailed understanding of AAV2 biology, most rAAV vectors generated today utilize the AAV2 ITRs in their vector designs. The sequences placed between the ITRs will typically include a mammalian promoter, gene of interest, and a terminator. Subtle differences in binding preferences, encoded in capsid sequence differences, can influence cell-type transduction preferences of the various AAV variants [21–23]. For example, AAV9 has a preference for primary cell binding through galactose [24], AAV2 uses the fibroblast/ hepatocyte growth factor receptor and the integrins αVb5 and α5b1; AAV6 utilizes the epidermal growth factor receptor; and AAV5 utilizes the platelet-derived growth factor receptor [25]. A deeper understanding of the AAV capsid properties has made the rational design of AAV vectors that display selective tissue/organ targeting possible, thus broadening the possible applications for AAV as a gene therapy vector. Pseudotyping of rAAV vectors is used to generate tropism-modified vectors. rAAV2 genomes can be packed into capsids derived from other AAV serotypes, thus narrowing or broadening the affinity of the new viral vector for specific cell types.

#### *Parvovirus Vectors: The Future of Gene Therapy DOI: http://dx.doi.org/10.5772/intechopen.105085*

AAV has been shown to be safe and effective in preclinical and clinical settings. Due to their oncogenic and immunogenic properties [26, 27], retroviral and adenoviral vectors may be associated with certain complications, but AAV has not been proven to cause any such pathological symptoms. Additionally, AAV possesses many desirable features like its ability to transduce nondividing cells [28, 29], broad host range [30], and the ability of the wild-type (wt) AAV genome to integrate site specifically into chromosome 19 in human cells [31, 32]. Besides, wt AAV has also been shown to possess antioncogenic properties [33]. AAV can infect not only actively dividing cells, but also quiescent cells, which makes it particularly valuable for many cell populations where viral and non-viral vectors are not sensitive to gene delivery, such as retinal cells and neuronal cells. The natural ability of AAV to infect quiescent cells has contributed to many significant advances in gene therapy, such as Luxturna (Spark Therapeutics) approved by the FDA for the treatment of Leber's congenital amaurosis [34].

In the past 20 years, the relevance of AAV vector-based therapy in clinical transformation has continued to increase, and it currently accounts for 8.1% of global gene therapy clinical trials. There are currently 17 gene therapies approved by the US FDA, including the AAV vector voretigene neparvovec rzyl (VN), which was developed by Spark Therapeutics in 2017 under the trade name Luxturna [34]. VN contains an AAV2 that wraps the RPE6 gene, which is used to treat biallelic RPE65-related retinal dystrophy, a rare genetic disease that leads to impaired visual function, declines with age, and ultimately leads to blindness. The second AAV-based gene therapy approved by the FDA in 2019 is Onasemogene abeparvovec xioi (OA), developed by AveXis under the trade name Zolgensma. OA uses AAV9 expressing a functional SMN1 transgene to treat type I spinal muscular atrophy (SMA1) in children under 2 years of age [35].

Most AAV successfully used in preclinical and clinical research is limited to natural capsid serotypes. The existence of neutralizing antibodies against AAV is still an important obstacle to systemic delivery [36]. These neutralizing antibodies interfere with the entry of AAV into target cells, intracellular transport and unpacking in the nucleus, thereby preventing transduction. Epidemiological studies have shown that neutralizing antibodies with different seropositivity rates can be found in 30–60% of the population. The most popular of these neutralizing antibodies is against AAV2, followed by AAV1. Another problem of AAV-mediated gene therapy is the size limit of the genome (4.7 kbp), including ITRs, leaving only a ∼4.5 kbp size space for the transferred gene. Engineered AAV can be designed through capsid modification, surface coupling and encapsulation to solve the limitations of natural AAV [37]. A common goal of AAV engineering is to avoid inactivation by neutralizing antibodies in the blood circulation after systemic administration. Another benefit of AAV engineering is to improve targeted delivery and activation by binding tissue-specific ligands to the capsid, surface coupling and encapsulating materials. Engineered AAV can also be used to overcome the limited genome size and combine multiple treatment modalities for multimodal therapy.
