**3. AAV-host cell interaction**

**1. Introduction**

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

may face.

**2. Adeno-associated virus**

Gene therapy is a novel promising approach for treating a spectrum of inherited and noninherited disorders by delivering therapeutic genes to specific organs or tissues. Of the viral vectors that have been used to date to deliver the gene of interest, the adeno-associated viral (AAV) vector appears to be the most safe and effective vehicle and has the ability to maintain long-term gene and protein expression following a single injection of the vector. Gene therapy studies using AAV vector have shown significant progress not only in animal models but also in human gene therapy with no known pathogenicity. Recently, the Food and Drug Administration (FDA) has approved a pioneering gene therapy protocol using an AAV vector for a rare form of childhood blindness, the first such treatment cleared in the United States for an inherited disease. While success has been achieved in this field targeting inherited disorders, however, clinical trials are yet to begin to see whether gene therapy has promise for treatment of non-inherited diseases. This chapter describes AAV biology, viral structure, and cell entry mechanisms, with special emphasis on AAV tissue tropism achieved by manipulating different serotypes and capsid engineering. This chapter also discusses successful application of the AAV vector for non-inherited disorders in animal models with particular reference to liver fibrosis, outlining advantages, disadvantages, and future challenges that this therapy

Adeno-associated virus (AAV) was discovered by Atchison et al. in 1965 from a pooled harvest of rhesus monkey kidney cell (RMK) cultures coinfected with simian adenovirus type 1 (SV15) [1]. This virus that could be observed as small DNA-containing particles was initially discovered as a contaminant of adenovirus preparations, and thus, it was named adeno-associated virus. However, AAV belongs to a genus of the parvoviruses, now known as dependoviruses [2]. AAV is replication defective and depends on a helper virus for effective and productive replication in mammalian cells. Generally, adenovirus or herpes viruses are considered to be the helper viruses for AAV to continue its life cycle. Early research on AAV has shown that this virus does not cause any disease in man even though it appears that it persists in humans along with its helper virus, particularly adenovirus [2]. In 1969, AAV was shown to possess several advantages in experimental systems including its small DNA genome of approximately 5 kb, packaging of plus and minus strands into individual particles, and most importantly, it is present as a defective virus [3]. During the first 20 years after its discovery, its genome structure, growth cycle, and latency were described. In the early 1980s, the genome sequencing of AAV serotype 2 (AAV2) was completed by Srivastava and colleagues [3]. This facilitated the generation of the first recombinant AAV vectors using AAV2 by the mid-1980s. Thereafter, studies using AAV were used for gene transfer in mammalian cell cultures. Subsequently, evidence of clinical safety has encouraged the researchers to use

The AAV2 is a non-enveloped virion with a genome consisting of a single-stranded DNA (ssDNA) which is enclosed by a spherical protein shell about 20 nm in diameter [5, 6], with

AAV vectors in clinical trials for various inherited disorders [4].

Heparan sulfate proteoglycan (HSPG) is the first identified primary receptor that AAV2 binds when infecting cells [6, 13]. The initial hypothesis was that the HSPG-binding site is located within the capsid protein VP3 [14], and this hypothesis was further supported by a mutational analysis performed by Wu and colleagues in year 2000 [11]. Wu et al. showed that there are two VP3 amino acid clusters of AAV2 that are involved in HSPG binding [11]. HSPG is not the only receptor type involved in AAV2 binding to a host cell, but there are one or more coreceptors which facilitate AAV cellular entry. Interestingly, αvβ5 integrin was identified as a coreceptor for internalization of AAV2 virions by Summerford and colleagues [15]. In cell studies, the chelating agent ethylenediaminetetraacetic acid (EDTA) was used to disrupt integrin function and results showed a notable reduction in AAV2 infection, suggesting that AAV2 uses αvβ5 integrin as a secondary receptor to mediate viral entry. Moreover, Qing and colleagues identified that human fibroblast growth factor receptor 1 (FGFR1) is also essential for viral entry into the host cell [16] and acts as a coreceptor for successful infection by AAV [6].

Although AAV2 is the most extensively studied serotype of AAV, there are several other AAV serotypes which have been evaluated for their binding characteristics to cellular receptors. It was recently shown that AAV serotypes 3 [17] and 13 (VR-942) [18] utilize HSPG as the primary cell surface receptor for cell entry, while AAV serotypes 1, 4, 5, and 6 [18–20] utilize N-linked and O-linked α2–3 and α2–6 sialic acids. AAV9 interacts with N-terminal galactose as the primary receptor [21] and also interacts with secondary coreceptors for facilitating cell entry, such as integrins [15, 22] FGFR1 [16], hepatocyte growth factor receptor (c-Met) [23], and laminin receptor [24]. Despite all these known pathways for AAV infection, no common primary receptor for all the AAV serotypes had been identified. Recently, Pillay and colleagues [25] used a library of mutagenized haploid HAP1 cells to create knockouts of nearly all nonessential genes in the human genome. This knockout library was exposed to recombinant AAV2-RFP (AAV2-red fluorescent protein), and a gene that was most significantly enriched in the screen was identified. This receptor named "AAV receptor (AAVR)" is characterized as a type I transmembrane protein which contains a MANSC domain, five polycystic kidney disease (PKD) domains, and a C6 region near the N terminus. These findings have been validated using an AAVR knockout cell line which demonstrated a resistant to infection by 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 AAVR as a universal primary receptor for all AAV serotype infection [6].

histocompatibility complex (MHC) class I pathway [36]. Although there are several studies that have investigated the nuclear entry of AAV, the mechanism by which AAV translocates into the nucleus is still unclear. Because AAV is a small virus with a diameter of around 20 nm, it has been suggested that the virion enters the nucleus using the nuclear pore complex (NPC) [37]. Furthermore, the nuclear entry of AAV is dependent on importin-β, a nuclear import protein that has been shown to play a key role in facilitating the binding of viral particles to host nuclei in other viral infectious pathways [38, 39]. Another study using single-point edge excitation sub-diffraction (SPEED) microscopy, a form of super-resolution imaging, to track single AAV particles revealed that approximately 17% of AAV particles were imported through the NPC successfully to the nucleus [40], reinforcing the importance of the NPC in AAV nuclear transfer. Interestingly, there is further evidence that nucleolin, a protein that shuttles between cytoplasm and nucleus, specifically binds to AAV capsid, which suggests that nucleolin may act as a nuclear receptor for AAV particles as well [41]. Upon entry into the nucleus, the ssDNA of AAV genome is converted to double-stranded DNA (dsDNA) using nuclear machinery of the target cells for transcription of the transgene [42]. The synthesis of second DNA strand has been considered as a rate-limiting factor for the onset and efficiency of transgene expression in ssAAV vectors [43]. As a result, second-generation AAV vectors with a dsDNA, also known as self-complementary AAV (scAAV) vectors, have been developed to improve the transduction and transcription efficiency. In the past decade, several studies have shown that new scAAV vectors provide safe, reliable, and organ-specific transduction both *in vitro* [44–46] and *in vivo* [46–49]. This suggests that the limitations associated with cell transduction using ssAAV genome can be overcome by the use of scAAV

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

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

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

vectors in gene therapy.

**5. AAV serotypes and tissue tropism**
