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

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

**Characteristics Tissue tropism**

Close homology to all serotypes except AAV 4,

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

AAV1 Shares 99% homology with AAV6 serotype [50] Liver, heart, skeletal muscle [52]

AAV6 Shares 99% homology with AAV1 serotype [50] Liver, heart, skeletal muscle [52] AAV7 Fast in targeting the tissues [52] Liver, skeletal muscle [52]

AAV12 Close homology to AAV4 serotype [50] Muscle, salivary glands [136]

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

AAV8 93% homology to AAV10 serotype [50] Heart, liver, brain, muscle (second most efficient

AAV10 93% homology to AAV8 serotype [50] Liver, heart, muscle, lung, kidney, uterus (with

AAV11 Close homology to AAV4 serotype [50] Muscle, kidney, spleen, lung, heart, stomach (with

AAV9 Fast in targeting the tissues [52] Liver, heart, brain, lung, skeletal muscle (serotype with

Liver, heart, muscle [52]

Lung, heart, liver, central nervous system [52, 134]

the broadest tissue tropism and most efficient in reaching

Heart, liver [52]

Liver [52]

the brain) [52]

serotype reaching the brain) [52]

pseudotype AAV2/10) [135]

pseudotype AAV2/11) [135]

and familial hypercholesterolemia [53].

**AAV serotype**

AAV2 The most commonly used serotype

Low transduction efficiency [52]

Slow in targeting the tissues [52]

AAV4 Close homology to AAV11 (82%) and AAV12

AAV5 The most divergent serotype (shares only 53–59% homology to other serotypes) Low transduction efficiency [52]

5, 11, and 12 [50]

AAV3 Low transduction efficiency

(79%) serotypes [50] Low transduction efficiency Slow in targeting the tissues [52]

> 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

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].

administration of 5α dihydrotestosterone in female mice prior to rAAV injection enhanced stable hepatocyte gene transfer to levels observed in male mice, suggesting rAAV vector

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

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

In addition, there is evidence to demonstrate the distinctly different patterns of persistence of rAAV-eGFP (enhanced green fluorescent protein) expression across the hepatic lobule in male and female mice. Female mice retained a predominantly perivenous pattern of expression, whereas male mice had shown an inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression [70]. Therefore, these sexually dimorphic patterns of genome persistence could have significant implications for the longterm therapeutic efficacy of rAAV-mediated gene transfer in man, particularly in the context

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

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

transduces hepatocytes via an androgen-dependent pathway [69].

of correction of liver functions showing metabolic zonation [70].

**9. Production and modification of AAV**

strand for transgene expression *in vivo* [6, 46, 82].

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 large prevalence of AAV neutralizing antibodies in humans.

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 transgene is to be either elicited or avoided.

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

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, administration of 5α dihydrotestosterone in female mice prior to rAAV injection enhanced stable hepatocyte gene transfer to levels observed in male mice, suggesting rAAV vector transduces hepatocytes via an androgen-dependent pathway [69].

In addition, there is evidence to demonstrate the distinctly different patterns of persistence of rAAV-eGFP (enhanced green fluorescent protein) expression across the hepatic lobule in male and female mice. Female mice retained a predominantly perivenous pattern of expression, whereas male mice had shown an inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression [70]. Therefore, these sexually dimorphic patterns of genome persistence could have significant implications for the longterm therapeutic efficacy of rAAV-mediated gene transfer in man, particularly in the context of correction of liver functions showing metabolic zonation [70].
