**1. Introduction**

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 may face.

a density of 1.41 g/cm<sup>3</sup>

**3. AAV-host cell interaction**

[6, 7]. The AAV genome is made up of 4675 nucleotides flanked by

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

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

inverted terminal repeats (ITRs). Each ITR is 145 nucleotides in length and forms a T-shaped hairpin structure by self-base pairing utilizing the first 125 nucleotides [3, 8]. Viral replication (Rep) and capsid (Cap) genes responsible for encoding four non-structural proteins, such as Rep40, Rep52, Rep68, and Rep78, and three structural proteins, such as VP1, VP2, and VP3, respectively, are located between the two ITR regions. The structural proteins, VP1, VP2, and VP3, are arranged in a ratio of 1:1:10 to form the icosahedral symmetrical shape of the virus [6, 9]. It has been reported that the VP1 protein is essential for infection [6, 10], whereas VP2 is the major protein responsible for nuclear transfer of the capsid proteins. Of note, the VP3 subunit is the most abundant protein in the capsid responsible for the binding of the virus to

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

cell surface receptors [6, 11] and viral particle formation in the host cell [12].

### **2. Adeno-associated virus**

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

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 a density of 1.41 g/cm<sup>3</sup> [6, 7]. The AAV genome is made up of 4675 nucleotides flanked by inverted terminal repeats (ITRs). Each ITR is 145 nucleotides in length and forms a T-shaped hairpin structure by self-base pairing utilizing the first 125 nucleotides [3, 8]. Viral replication (Rep) and capsid (Cap) genes responsible for encoding four non-structural proteins, such as Rep40, Rep52, Rep68, and Rep78, and three structural proteins, such as VP1, VP2, and VP3, respectively, are located between the two ITR regions. The structural proteins, VP1, VP2, and VP3, are arranged in a ratio of 1:1:10 to form the icosahedral symmetrical shape of the virus [6, 9]. It has been reported that the VP1 protein is essential for infection [6, 10], whereas VP2 is the major protein responsible for nuclear transfer of the capsid proteins. Of note, the VP3 subunit is the most abundant protein in the capsid responsible for the binding of the virus to cell surface receptors [6, 11] and viral particle formation in the host cell [12].
