**5. AAV serotypes and tissue tropism**

almost all AAV serotypes, whereas restoring recombinant AAVR gene in the AAVR knockout cells restored the ability of AAVs for successful infection. Furthermore, AAVR gene knockout mice demonstrated robust resistance to AAV9 infection. This important discovery implicates

The major cell entry mechanism for AAV is via endocytosis utilizing clathrin-coated pits, although other minor mechanisms are possibly involved in this process. However, these alternative minor mechanisms are yet to be confirmed [6]. Upon AAV binding to its cell surface receptors, it stimulates intracellular signaling pathways, which in turn stimulates internalization of AAV. This phenomenon can be clearly explained using the mechanisms reported for AAV2 host cell interaction. It was shown that attachment of AAV2 to HSPG and αVβ5 integrin resulted in the activation of Rac1, an intracellular small guanosine triphosphate (GTP)-binding protein, and phosphoinositide 3-kinase (PI3K) in HeLa cells within 5 minutes of AAV2 infection [26]. Furthermore, inhibition of Notch1 by siRNA, a transmembrane receptor known to be involved in the activation of Rac1 and PI3K, was reported to decrease cell transduction by AAV2 [27], suggesting that the Rac1-PI3K pathway is necessary to initiate endocytosis of AAV2. Direct injection of AAV into the cytoplasm and nucleus of cells results in a significant lower infection rate than cells that are simply exposed to virus [28], suggesting that the processing of AAV virion through endosomal compartments is a critical initiating

In addition, transduction efficiency of AAV is largely dependent on the endosomal pH. Changing the pH to acidic (pH 4–6) inside the endosomal compartment facilitates transduction of AAV, whereas blocking acidification during endosomal processing decreases the rate of transduction [29–31]. Also, the application of different classes of proteasome inhibitors such as tripeptidyl aldehydes and N-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL) and the anthracycline compounds such as doxorubicin increases the rate of viral translocation to the nucleus [32]. Furthermore, LLnL appears to increase AAV2 capsid ubiquitination that results in augmented gene transfer in different cell types [33], suggesting a mechanism by which these inhibitors increase transduction may be related to ubiquitination. AAV must exit from the endosome first before translocating to the nucleus. Prior to escape from the endosome, AAV undergoes a conformational change leading to the exposure of the unique N-terminal ends of VP1 and VP2, which contains a domain of phospholipase A2 (PLA2) [34], an enzyme that breaches the endosomal membrane and thereby facilitates efficient endosomal escape of viral particles. Upon endosomal escape, AAV enters the nucleus as an intact particle [28] and uncoating then occurs inside the nucleus. However, nuclear transport of AAV is a slow process, approximately only 1–2% of internalized AAV enters and expresses in the nucleus, and the whole entry process takes about 2–13 h [35]. Thus, most viral particles which fail to

The viral particles that fail to translocate into the nucleus are eventually degraded by host proteasomes in the cytoplasm and presented as antigen to cytotoxic T cells via the major

the AAVR as a universal primary receptor for all AAV serotype infection [6].

**4. Cell entry mechanism of AAV**

142 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

step for transduction following endocytosis.

translocate are located outside or away from the nucleus.

To date, a total of 12 naturally occurring AAV serotypes have been discovered from both human and non-human primates (**Table 1**). These serotypes are able to infect cells of diverse tissue types. Interestingly, the tissue specificity is determined by the capsid serotype. The existence of a variety of serotypes with different infectivity rates and tissue specificity makes AAV one of the most promising candidates in gene therapy research. By development of different AAV pseudotypes, researchers have been able to obtain unique cellular tropism and high transduction efficiency. All AAV serotypes share at least 50% sequence homology. However, serotype AAV5 has the most divergent amino acid capsid sequence, and AAV4 also shows a considerable degree of divergence [50]. Surprisingly, this sequence diversity between serotypes is not scattered but primarily located in the looped out domains of the capsid protein [51]. However, comparative studies of AAV serotypes found that this sequence variability may not be responsible for the differences in infectivity rates and tissue specificity. AAV serotype 2 is most widely used in gene therapy research. Several studies have investigated gene expression and tropism *in vivo* mediated by different AAV serotypes and identified that they differ broadly in transduction efficacies and tissue tropism. A comparative study of AAV serotypes 1–9 mediated transgene expression after systemic


**6. Molecular engineering of AAV capsid**

prior to commencing clinical studies.

There are several challenges for AAV serotypes to exert their therapeutic potential in target organs including the need for high vector doses for efficient delivery, pre-existing antiviral immunity in the host, and the lack of cell type-specific tropism leading to off-target transduction [6]. One way to overcome these limitations is to randomly generate capsid mutants from a library to extend the capability of the traditional AAV vector by increasing its cell transduction efficiency for specific cell types and its ability to escape from antibody neutralization.

Adeno-Associated Virus (AAV)-Mediated Gene Therapy for Disorders of Inherited…

http://dx.doi.org/10.5772/intechopen.80317

One approach used to create a mutant library is DNA shuffling, a strategy in which the open reading frame of capsid genes of different AAV serotypes is fragmented by nucleases. This is followed by random ligation, resulting in new and random combinations of capsid sequences. These new molecular-enhanced AAV vectors exhibit a broad range of cell tropism with numerous functional differences between chimeras and their parent serotypes. Consequently, there is potential to produce unlimited numbers of new AAV variants with novel gene delivery properties. This method of AAV capsid engineering was first described in 2008 by Grimm and colleagues [54] and has become a commonly used technique over the years. More recently, Lisowski and colleagues utilized a humanized mouse model to perform serial selection using a human-specific replication competent viral library composed of DNA-shuffled AAV capsids. After four rounds of selection, they identified a novel chimeric capsid variant composed of five different parental AAV capsids [55]. Of these, LK-03, which efficiently transduced human primary hepatocytes both *in vitro* and *in vivo*, was found to be a human liver cell-specific AAV serotype [55]. This study has opened up a new avenue to validate therapeutic potential of an AAV capsid variant in preclinical studies using human primary cell xenotransplanted models

In addition, a study using *in silico* ancestral sequence reconstruction (ASR) of AAV capsid protein generated nine functional putative ancestral AAVs. In this study, Zinn and colleagues also identified Anc80, the predicted ancestral sequence of the widely used AAV serotypes 1, 2, 8, and 9 and showed that Anc80 is a highly potent *in vivo* gene therapy vector compared to AAV2 and AAV8 for targeting liver, muscle, and retina in mice [56]. Nevertheless, Anc80 demonstrated a high stability and no toxicity in several safety studies carried out in mice. This synthetic viral vector has been evaluated in non-human primates (rhesus macaques), which demonstrated a superior expression of Anc80 in monkey liver following Anc80 administration compared to control monkeys injected with AAV8. Hence, future studies may also rely

It has been shown that AAV viral proteins cause a minimal immunogenic response, and at the same time, it can yield prolonged expression of therapeutically relevant genes/proteins. Also, when comparing to the other potential viral vectors such as lentiviral vectors, AAV possesses a reduced proinflammatory risk and has been considered as one of the most promising gene

on the use of Anc80, in particular for liver-directed gene therapy studies.

**7. AAV as a safe vector in gene therapy**

**Table 1.** Characteristics and tissue tropism of AAV serotypes in the mouse.

tail vein injection in mice showed that each AAV serotype profoundly differs in its ability to transduce organs, with AAV9 having the highest and fastest onset of transgene expression, highest viral genome copies, and the broadest tissue tropism, as determined by luciferase images [52]. Conversely, AAV3 and AAV4 are the slowest in targeting tissues, and among all the serotypes, AAV2, 3, 4, and 5 have the lowest transduction efficiency. The liver is the most common organ transduced by nearly all AAV serotypes with AAV7 and AAV9 showing the strongest tropism. Moreover, AAV9 is the most efficient serotype in reaching the heart and brain, followed by AAV4 and AAV8, respectively [52]. Of note, AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than the other AAV serotypes, and therefore, this serotype has been developed to use as a gene therapy vector for hemophilia A and familial hypercholesterolemia [53].
