**9. Production and modification of AAV**

transfer vectors for *in vivo* gene therapy [57]. However, in some experimental settings, it was reported that immune responses generated by AAV administration appear to compromise the outcomes of AAV-mediated gene therapy. Thus, several factors may determine the occurrence of immune responses against the AAV proteins, including the route of administration, dose, serotype, host species, transgene and expression cassettes, and pre-existing immunity to AAV [6, 58]. It has been suggested that AAV activates mouse and human plasmacytoid DCs to produce type 1 interferon via a TLR9-MyD88 pathway, resulting in induction of adaptive immune CD8+ T cell responses to AAV capsid and the transgene [58]. In addition, different administration routes for AAV2-mediated ocular gene therapy induced varying immune responses. For instance, intravitreal administration of an AAV2 vector, which led to transduction of the inner retina, triggered a humoral immune response to AAV2 capsid; however, no effect was observed following subretinal administration and subsequent repeated injections [59]. Animal studies have suggested that the presence of neutralizing antibodies could compromise AAV transduction *in vivo* following systemic administration [60, 61]. These findings are potentially important for translation of AAV gene therapy from animal studies to clinical trials due to the

Due to natural exposure to wild-type AAV early in life, a significant proportion of human population have humoral immunity to the AAV capsid, primarily AAV1, 2, 3, and 5 [62, 63]. Of note, among the most commonly used AAV vectors, the most prevalent anti-AAV antibodies in humans are AAV2 followed by anti-AAV antibodies to AAV1 [64], while the least prevalent are for AAV7 and AAV8. It has been shown that rAAV vectors, including serotypes 1, 2, and 5 can transduce dendritic cells (DCs) and generate immune responses to transgene products [65, 66]. Interestingly, another study, which evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors using a hypersensitive autoimmune mouse model, revealed that unlike AAV1 vectors, AAV8 vectors were unable to transduce dendritic cells (DCs) and elicit transgene-specific immune responses efficiently, resulting in induction of immune tolerance to transgene products [67]. Different properties of these vectors imply tremendous potential in different applications, where an immune response to

Recombinant AAV vector transduction efficiency clearly depends on the gender. This fact has been specifically shown in the liver and the brain in murine models. A study carried out by Maguire and colleagues has shown that the vector transduction efficiency using AAV serotype 9 was found to be different in the brain and the liver between male and female mice [68]. This study revealed a higher transgene expression in the brain of females compared with male mice, whereas a higher transgene expression was observed in the liver of male mice compared with female mice. In line with this study, Davidoff and colleagues revealed that when compared with female mice, transgene expression after liver-targeted delivery of AAV2 and AAV5 particles was 5- to 13-fold higher in male mice [69]. In addition, they found that transduction efficiency was dramatically reduced by castration in male mice, whereas oophorectomy in female mice did not significantly influence rAAV transduction [69]. Moreover,

large prevalence of AAV neutralizing antibodies in humans.

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

transgene is to be either elicited or avoided.

**8. AAV vector transduction efficiency—male versus female**

The AAV serotype 2 was the first AAV vector used for gene transfer applications. This particular vector was chosen primarily because of its broad tropism, efficient transduction with stable and long-term transgene expression with minimal inflammation, and immune responses in a number of organs, such as the brain [71], retina [72], and skeletal muscles [73]. Liver is the other major organ which is targeted for rAAV2 gene delivery strategy because hepatocytes are easily accessible to vectors injected into the circulation through large pores in liver capillaries. Although results in the liver have been less consistent, a number of studies demonstrate a successful transduction of rAAV2 vector with persistent transgene expression in the liver using a single dose [74], and approximately 5% of hepatocytes were transduced following rAAV2 vector injection [75]. Of note, a study which was undertaken by Snyder and colleagues provided the most impressive results by achieving sustained and therapeutic levels of factor IX in hemophilia B, with no associated toxicity in both canine and murine models [75, 76].

The discovery of novel strategies for pseudotyping, recombination of AAV constructs into capsids of alternative serotypes, and the development of scAAV vectors which effectively alter tissue tropisms with enhanced transduction efficiency [77] has opened up new avenues to produce more attractive vectors for use in clinical applications including hemophilia B, Parkinson's disease, and rheumatoid arthritis [78]. Among all novel recombinant AAV serotypes, AAV2 genome construct pseudotyped with capsid 8 (AAV2/8) is one of the most efficient vectors for hepatic gene transfer. In addition, it has greater liver transduction efficiency, with fourfold more genomes per transduced cell, when compared with other pseudotyped vectors [6, 79]. Moreover, it has an excellent transduction rate (95%) in hepatocytes of the mouse liver via intraportal vein injection [80]. In line with this, the development of scAAV vectors further enhances the transduction efficiency to the liver [81], suggesting that the conversion of single-stranded AAV genome into double-stranded form for gene therapy studies appears to be beneficial since this procedure can avoid the need to assemble second DNA strand for transgene expression *in vivo* [6, 46, 82].

The most widely used method to produce and purify recombinant AAV particles for preclinical applications is the triple transfection method using HEK293 cells, which requires the use of an

to be the leading vector for future gene therapy studies [88]. Unlike recombinant adenoviral vectors which yield high initial gene expression that diminishes rapidly due to immune clearance, the AAV vector-based gene expression is persistent. In addition, as AAV vectors were derived from a parental virus with no known pathogenesis which is replication defective, they do not carry a risk of infecting patients with a pathogenic wild type virus. In addition, AAV vectors mediate a minimal cell-mediated immune response, which is favorable for the persistence gene transduction to the host cells. At the same time, AAV-based vectors are able to transduce a wide range of host cells including both dividing and non-dividing cell types [88, 89]. A prominent disadvantage associated with AAV compared to the other viral vectors is its small packaging size, which limits the size of the transgene to be delivered using the vector. However, novel molecular engineering methods have the potential to overcome these limitations, and thus, genetically engineered AAV is poised to become the leading vector for future gene therapy in humans.

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

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

Many studies have explored the therapeutic potential of these engineered AAV vectors for a number of inherited disorders. After several decades of experimental studies, the first successful human gene therapy protocol using AAV serotype 1 vector was approved in 2012 by the European Commission (EU) for the treatment of patients with lipoprotein lipase deficiency (LPLD), an extremely rare genetic disorder [90]. This was a milestone achievement for researchers who have been working to develop successful gene therapy protocols for inherited human disorders. The therapy was introduced under the trade name Glybera® (alipogene tiparvovec) by UniQure. However, after 5 years of the launch of the world's first approved gene therapy, UniQure has not renewed its EU license in 2017 and ceased to produce Glybera for use because of the expensive nature of the treatment protocol [91]. However, it was unfortunate that UniQure has discontinued its production despite the first LPLD patient treated with alipogene tiparvovec showing improvement of quality of life without abdominal pain and pancreatitis attacks for 18 months [92]. UniQure, however, has endeavored to develop

The most common clinical trials based on AAV therapy in recent years have been in hemophilia B, a blood clotting disorder caused by a defect in the gene encoding coagulation Factor IX (FIX), leading to a deficiency of FIX. The only treatment available for this disease is lifelong intravenous infusion of FIX concentrates. Although this treatment is effective as a preventive medicine, it is not curative. In addition, the treatment is invasive, inconvenient, and very expensive, thus not affordable for most patients with hemophilia B, resulting in a reduction in life expectancy for those patients with a severe bleeding phenotype [93]. Similar to the FIX concentrates, there are clotting formulations with longer half-life which represents a major advance but still require lifelong intravenous administration. Robust preclinical results using AAV-based therapy in two murine [74, 94] and three canine models of hemophilia B [95–97] demonstrated long-term expression of FIX, with no significant liver toxicity and with no FIX-specific antibodies detected following muscle- or liver-directed injections. A follow-up study demonstrated an induction of immune tolerance in mice after hepatic gene transfer by rAAV expressing human FIX (rAAV-hFIX), which is mediated by regulatory CD4+ T cells, resulting in suppression of human FIX antibody formation [6, 98]. Based on the results from

**11. Gene therapy using AAV vectors for inherited disorders**

gene therapy for hemophilia B.

**Figure 1.** Schematic representation of the assembly of AAV2 genome pseudotyped with liver-specific AAV serotype 8 and liver-specific promoters in HEK293 cells. Liver-specific rAAV2/8-ACE2 viral particles are produced by transfecting HEK293 cells with rep2/cap8 plasmid, Ad helper plasmid, and a plasmid carrying AAV2 inverted terminal repeat-ACE2 cassette with liver-specific promoters. Recombinant AAV2/8-ACE2 viral particles are purified from cell homogenate 48–72 h post transfection, followed by assessment of AAV quality, genome titer, infectious and transducing properties, and integrity of the packaged AAV genome [87].

AAV replication and capsid plasmid that provides Rep78, Rep68, Rep52, and Rep40 proteins necessary for vector genome replication and VP1, VP2, and VP3 capsid proteins, the vector DNA plasmid with the inverted terminal repeat-transgene cassette, as well as the adenovirus (Ad) helper plasmid [83, 84]. In addition, HEK293 cells have been engineered to provide adenovirus helper genes *in trans* such as E1a and E1b55k for AAV assembly. The key advantage of this method is that AAV particles can be efficiently made with genes supplied by Ad helper and HEK293 cells without the need to use replication competent adenovirus [84] (**Figure 1**). In addition, to improve tissue tropism, the AAV genomes can be pseudotyped with a desired capsid protein. Following 48–72 h transfection, the cell homogenate is purified, followed by the assessment of AAV quality control including genome titer [85], infectious and transducing properties, and integrity of the packaged AAV genome [86]. This is schematically illustrated in **Figure 1** where AAV2 genome is pseudotyped with capsid 8 (AAV2/8) to increase liver specificity [87].

## **10. Pros and cons of AAV gene therapy**

A successful gene therapy approach should deliver an appropriate amount of a therapeutic gene into the target tissue without substantial toxicity while achieving long-term gene expression. Of all currently available viral vectors including retroviral, lentiviral, adenoviral, and AAV vectors, the AAV is a unique non-pathogenic viral vector with broad tissue tropism and has the potential to be the leading vector for future gene therapy studies [88]. Unlike recombinant adenoviral vectors which yield high initial gene expression that diminishes rapidly due to immune clearance, the AAV vector-based gene expression is persistent. In addition, as AAV vectors were derived from a parental virus with no known pathogenesis which is replication defective, they do not carry a risk of infecting patients with a pathogenic wild type virus. In addition, AAV vectors mediate a minimal cell-mediated immune response, which is favorable for the persistence gene transduction to the host cells. At the same time, AAV-based vectors are able to transduce a wide range of host cells including both dividing and non-dividing cell types [88, 89]. A prominent disadvantage associated with AAV compared to the other viral vectors is its small packaging size, which limits the size of the transgene to be delivered using the vector. However, novel molecular engineering methods have the potential to overcome these limitations, and thus, genetically engineered AAV is poised to become the leading vector for future gene therapy in humans.
