**12. Gene therapy for non-inherited disorders**

animal studies, the world first clinical trial using rAAV2-hFIX vector in humans via intramuscular route has been conducted [99]. The results indicated that the transduction of muscle tissue was successful; however, circulating plasma FIX levels in all patients were less than the required level for a therapeutic effect (<2% of normal). In a subsequent clinical study, the delivery target was switched to the liver, the normal site of FIX synthesis. Although rAAV2 mediated hFIX gene transfer to the liver-mediated therapeutically relevant expression levels

Recent study by Nathwani and colleagues demonstrated the AAV8 serotype as a more effective vector for liver-directed hemophilia B gene therapy [101]. In this study, six severe hemophilia B patients received a single injection of pseudotyped AAV2/8-hFIX vector at three escalating doses (high, intermediate and low), with two patients per dose and no immunosuppressive was given. Patients were subsequently followed for up to 16 months. All patients have achieved AAV2/8-mediated expression of FIX at above the therapeutic threshold, ranging between 2 and 11% of normal levels, and the increase in FIX serum level was dosedependent. Four out of six patients discontinued their prophylactic treatment with hFIX concentrates without having spontaneous hemorrhage, whereas the other two patients continued to receive hFIX concentrates but extended the interval between hFIX treatments. This was the first liver-directed AAV gene therapy trial to show sustained therapeutic FIX levels and improved clinical outcomes in patients with hemophilia B. However, in patients who received the highest dose of vector, T cell-mediated clearance of AAV-transduced hepatocytes was observed, with associated elevation of liver enzyme levels. This response has been overcome

Nathwani and colleagues later conducted a follow-up study to evaluate the long-term safety and efficacy of AAV2/8-hFIX therapy in the same cohort of hemophilia B patients [93]. Of note, this monitoring study also included addition of four new patients, each of whom received the high dose of vector. Consistent with their previous findings, a single intravenous injection of vector resulted in an increase in plasma FIX activity from less than 1% to sustained level of up to 6% of the normal value in all 10 patients, and this remained stable for up to a period of 4 years. Additionally, substantial clinical improvements were achieved in all patients, including significant reductions in number of spontaneous hemorrhage and annual number of prophylactic treatment with FIX concentrates. Not surprisingly, there was a dose-dependent, asymptomatic increase in both the serum alanine transaminase (ALT) level and increase in anti-AAV capsid neutralizing antibody level, which led to a gradual decline in FIX levels, suggesting transduced hepatocyte destruction. There was a transient increase of ALT levels in all patients which resolved with administration of a single course of prednisolone, after which

A recent clinical trial completed using ssAAV vector consisted of a bioengineered capsid, liverspecific promoter, and FIX Padua (FIX-R338L) in 10 men with hemophilia B who had FIX coagulant activity of 2% or less of the normal also showed a success with no serious adverse events during or after vector infusion [102]. These patients were followed up to 492 days (16 months). The results showed that 8 of 10 patients did not require the regular treatment with FIX concentrates, and bleeding episodes were not reported in 9 patients after the vector treatment. Overall, there was a significant reduction in annual bleeding rate in patients treated with AAV-FIX-R338L. Although there were two patients who developed asymptomatic increase in liver

by a short course of glucocorticoids, without the loss of hFIX expression.

no recurrent elevation of serum ALT in patients was observed.

[100], the expression persisted for less than 8 weeks.

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

There have been many advances in identification of the mechanisms involved in chronic organ damage which opened up avenues for gene therapy studies [108]. While a plethora of preclinical and clinical studies over past several decades has focused on developing gene therapy for inherited disorders, despite several preclinical studies in animal models, there have been only a few clinical trials that have been undertaken to investigate therapeutic efficacy of gene therapy for non-inherited diseases. A recent study shows that telomerase expression using AAV9 vectors exerts therapeutic effects in a mouse model of pulmonary fibrosis [109]. This therapy targeted idiopathic pulmonary fibrosis. It is known that telomeres act as protective structures at the ends of chromosomes and the presence of short telomeres has been shown to be one of the causes for disease development. In this condition, telomeres become too short, resulting in the cessation of cell division which in turn leads to cell apoptosis. Telomerase is an enzyme that can restructure the telomeres length, and Povedano and colleagues developed a treatment using AAV serotype 9 to deliver telomerase to correct the short telomeres. As AAV9 preferentially targets regenerative alveolar type II cells (ATII), AAV9-Tert-treated mice show improved lung function with reduced inflammation and fibrosis at 1–3 weeks after vector treatment. It is of interest to note that pulmonary fibrosis either improved or disappeared at 8 weeks of gene therapy. AAV9-Tert treatment lead to longer telomeres and increased proliferation of ATII cells, as well as lower DNA damage, apoptosis, and senescence.

2 (ACE2), which breaks down the potent profibrotic octapeptide, angiotensin II (Ang II) to an antifibrotic heptapeptide, angiotensin-(1–7) (Ang-(1–7)) [120, 121]. Evidence from experimental animal studies showed that recombinant human ACE2 (rhACE2) is beneficial for prevention of hypertension in cardiovascular disease [122] and to improve kidney function in diabetic nephropathy [123]. Interestingly, rhACE2 was well tolerated by a group of healthy human volunteers in a phase 1 clinical trial, without exerting any unwanted cardiovascular side effects [124]. There is one study that reported therapeutic effects of recombinant ACE2 in experimental liver fibrosis, in which liver injury was surgically induced by cholestasis or by hepatotoxic carbon tetrachloride injection [125]. They demonstrated that recombinant ACE2 significantly reduced hepatic fibrosis in both animal models of liver disease [125]. However, a major drawback of this systemic approach is that the treatment inevitably produces offtarget effects, which in many cases are undesirable. Thus, there are several disadvantages with systemic administration of recombinant ACE2. This includes daily injections of ACE2, a procedure that is invasive in a clinical setting and expensive approach with unwanted effect on blood pressure regulation [125, 126]. To circumvent this problem, an ideal approach would be to increase tissue-specific ACE2 levels in the target organ. Thus, organ-specific increased ACE2 activity using a liver-specific recombinant AAV vector is expected to produce therapeutic effects confined to the targeted organ while minimizing unwanted off-target effects.

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

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

In addition to the use of liver-specific capsid serotype, specificity can be further enhanced by engineering the vector with ACE2 gene under the transcriptional control of a strong liver-specific promoter, apolipoprotein E/human α1-antitrypsin. Studies published by our laboratory used a pseudotyped liver-specific AAV vector (rAAV2/8) for preclinical evaluation and found that hepatic overexpression of murine ACE2 gene delivered into the mice lasted for up to 6 months following a single intraperitoneal injection [87]. We then treated mice with a range of liver disease models, which included biliary fibrosis induced by bile duct ligation (BDL), toxic injury induced by carbon tetrachloride (CCl4) injections, and fatty liver-associated liver fibrosis induced by feeding methionine- and choline-deficient (MCD) diet using a single intraperitoneal injection of rAAV2/8-ACE2 [87]. The treatment produced a major increase in ACE2 expression and protein activity, which was confined to the liver without affecting other major organs. Unlike inherited disorders, for example, hemophilia B where a relatively low level of transgene expression in the liver may be sufficient for subsequent small increases in FIX levels in the blood [48, 81], the magnitude of the expression of transgene required for therapeutic intervention in non-inherited disease may be substantially higher. This, in turn, may pose a challenge for gene therapy researchers. Interestingly, however in our liver-targeted therapeutic approach with rAAV2/8-ACE2, we found that increased hepatic ACE2 expression reduced hepatic level of profibrotic Ang II by more than 50% compared to those treated with a control vector that carried human serum albumin (rAAV2/8-HSA) [87]. A reduction of Ang II, which was accompanied by increases in hepatic levels of antifibrotic Ang-(1–7) peptide, resulted in a marked reduction in inflammatory cytokine expression, leading to a profound reduction in hepatic fibrosis in all three models (**Figure 2**) [87]. These studies with short-term animal models have been further validated to provide evidence that in long-term animal models of biliary fibrosis and fatty liver disease, which produce hepatic lesions more comparable to those seen in patients with such diseases, a single intraperitoneal injection of rAAV2/8-ACE2 caused a profound reduction in hepatic fibrosis (**Figure 3**). In marked contrast to other studies using

AAV vector-derived cardiac gene therapy is emerging as an entirely new platform to treat cardiac disorders [110]. AAV gene therapy for heart failure have been validated in preclinical studies using animal models, and the vast majority of these approaches have been undertaken to improve calcium handling by cardiomyocytes. The therapeutic protein used in the majority of these studies was sarcoplasmatic calcium ATPase (SERCA2a). Based on the positive preclinical findings, the first clinical trial (CUPID trial: calcium upregulation by percutaneous administration of gene vector in cardiac disease, NCT02346422) was carried out to deliver SERCA2a using AAV serotype 1 vector to treat patients with advanced heart failure [111, 112]. The outcome of this phase 1 trial was successful with no adverse events and was progressed to phase 2a study, providing promising outcomes with significantly low rate of adverse events. However, the results of phase 2b clinical trial (CUPID2b trial, NCT01643330) using the same vector were disappointing with no significant change between the treatment group and the placebo group [113]. This has led to the cessation of patient recruitment for two additional trials using AAV1. SERCA2a [110]. Interestingly, there are two new upcoming trials aimed to deliver S100A1 with an AAV9 vector and a constitutively active form of the protein phosphatase 1 inhibitors, I1c, with a chimeric capsid with AAV2 and AAV8 serotypes [114, 115]. In addition, AAV1, AAV6, and AAV9 have emerged as the most promising AAV serotypes for cardiac gene transfer, which provides hopes for successful gene therapy approaches to treat heart failure in the future.

AAV-mediated gene therapy approaches to treat neuropathic pain in rodents have also been reported [116]. Fischer and colleagues have shown that administration of rAAV expressing Ca2+ channel-binding domain 3 (CBD3) gene significantly reduced pain behavior such as hyperalgesia after touch with a pin or sensitivity to acetone stimulation in animal models of inflammatory and neuropathic pain [117]. Another study using AAV9 vector encoding short hairpin RNA (shRNA) against vanilloid receptor 1 (TRPV1), which is an important target gene for acute pain, demonstrated that the therapy attenuated nerve injury-induced thermal allodynia (increased response of neurons) 10–28 days after treatment in a mouse model of spared nerve injury (SNI) [118]. These results provide positive evidence to encourage gene therapy researchers to develop AAV vector-based treatments for patients with chronic/diabetic neuropathic pain.

Considerable progress has been made in gene therapy approach to treat chronic liver fibrosis. Although angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) are widely used as treatments in patients with hypertension, they have been trialed in patients with chronic liver disease; however, the outcomes were not convincing mainly because they produce adverse systemic side effects [119]. Because of the lack of medical treatments, liver transplantation has inevitably become the only option for patients with end stage liver disease, resulting from chronic hepatic fibrosis and/or cirrhosis. Moreover, increasing incidence of chronic liver disease, lack of donor organs, post-transplantation complications, and the high cost in liver transplantation mean that there is a major need to discover and formulate specific, effective, safe, and inexpensive novel therapies for liver fibrosis/cirrhosis.

One possible approach to circumvent this is to develop organ-targeted antifibrotic strategies. Studies from our laboratory suggested that one possible target is the "alternate axis" of the renin-angiotensin system (RAS), comprising its key enzyme angiotensin-converting enzyme 2 (ACE2), which breaks down the potent profibrotic octapeptide, angiotensin II (Ang II) to an antifibrotic heptapeptide, angiotensin-(1–7) (Ang-(1–7)) [120, 121]. Evidence from experimental animal studies showed that recombinant human ACE2 (rhACE2) is beneficial for prevention of hypertension in cardiovascular disease [122] and to improve kidney function in diabetic nephropathy [123]. Interestingly, rhACE2 was well tolerated by a group of healthy human volunteers in a phase 1 clinical trial, without exerting any unwanted cardiovascular side effects [124]. There is one study that reported therapeutic effects of recombinant ACE2 in experimental liver fibrosis, in which liver injury was surgically induced by cholestasis or by hepatotoxic carbon tetrachloride injection [125]. They demonstrated that recombinant ACE2 significantly reduced hepatic fibrosis in both animal models of liver disease [125]. However, a major drawback of this systemic approach is that the treatment inevitably produces offtarget effects, which in many cases are undesirable. Thus, there are several disadvantages with systemic administration of recombinant ACE2. This includes daily injections of ACE2, a procedure that is invasive in a clinical setting and expensive approach with unwanted effect on blood pressure regulation [125, 126]. To circumvent this problem, an ideal approach would be to increase tissue-specific ACE2 levels in the target organ. Thus, organ-specific increased ACE2 activity using a liver-specific recombinant AAV vector is expected to produce therapeutic effects confined to the targeted organ while minimizing unwanted off-target effects.

treatment lead to longer telomeres and increased proliferation of ATII cells, as well as lower

AAV vector-derived cardiac gene therapy is emerging as an entirely new platform to treat cardiac disorders [110]. AAV gene therapy for heart failure have been validated in preclinical studies using animal models, and the vast majority of these approaches have been undertaken to improve calcium handling by cardiomyocytes. The therapeutic protein used in the majority of these studies was sarcoplasmatic calcium ATPase (SERCA2a). Based on the positive preclinical findings, the first clinical trial (CUPID trial: calcium upregulation by percutaneous administration of gene vector in cardiac disease, NCT02346422) was carried out to deliver SERCA2a using AAV serotype 1 vector to treat patients with advanced heart failure [111, 112]. The outcome of this phase 1 trial was successful with no adverse events and was progressed to phase 2a study, providing promising outcomes with significantly low rate of adverse events. However, the results of phase 2b clinical trial (CUPID2b trial, NCT01643330) using the same vector were disappointing with no significant change between the treatment group and the placebo group [113]. This has led to the cessation of patient recruitment for two additional trials using AAV1. SERCA2a [110]. Interestingly, there are two new upcoming trials aimed to deliver S100A1 with an AAV9 vector and a constitutively active form of the protein phosphatase 1 inhibitors, I1c, with a chimeric capsid with AAV2 and AAV8 serotypes [114, 115]. In addition, AAV1, AAV6, and AAV9 have emerged as the most promising AAV serotypes for cardiac gene transfer, which provides hopes for successful gene therapy approaches to treat heart failure in the future.

AAV-mediated gene therapy approaches to treat neuropathic pain in rodents have also been reported [116]. Fischer and colleagues have shown that administration of rAAV expressing Ca2+ channel-binding domain 3 (CBD3) gene significantly reduced pain behavior such as hyperalgesia after touch with a pin or sensitivity to acetone stimulation in animal models of inflammatory and neuropathic pain [117]. Another study using AAV9 vector encoding short hairpin RNA (shRNA) against vanilloid receptor 1 (TRPV1), which is an important target gene for acute pain, demonstrated that the therapy attenuated nerve injury-induced thermal allodynia (increased response of neurons) 10–28 days after treatment in a mouse model of spared nerve injury (SNI) [118]. These results provide positive evidence to encourage gene therapy researchers to develop

Considerable progress has been made in gene therapy approach to treat chronic liver fibrosis. Although angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) are widely used as treatments in patients with hypertension, they have been trialed in patients with chronic liver disease; however, the outcomes were not convincing mainly because they produce adverse systemic side effects [119]. Because of the lack of medical treatments, liver transplantation has inevitably become the only option for patients with end stage liver disease, resulting from chronic hepatic fibrosis and/or cirrhosis. Moreover, increasing incidence of chronic liver disease, lack of donor organs, post-transplantation complications, and the high cost in liver transplantation mean that there is a major need to discover and formulate specific, effective, safe, and inexpensive novel therapies for liver fibrosis/cirrhosis. One possible approach to circumvent this is to develop organ-targeted antifibrotic strategies. Studies from our laboratory suggested that one possible target is the "alternate axis" of the renin-angiotensin system (RAS), comprising its key enzyme angiotensin-converting enzyme

AAV vector-based treatments for patients with chronic/diabetic neuropathic pain.

DNA damage, apoptosis, and senescence.

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

In addition to the use of liver-specific capsid serotype, specificity can be further enhanced by engineering the vector with ACE2 gene under the transcriptional control of a strong liver-specific promoter, apolipoprotein E/human α1-antitrypsin. Studies published by our laboratory used a pseudotyped liver-specific AAV vector (rAAV2/8) for preclinical evaluation and found that hepatic overexpression of murine ACE2 gene delivered into the mice lasted for up to 6 months following a single intraperitoneal injection [87]. We then treated mice with a range of liver disease models, which included biliary fibrosis induced by bile duct ligation (BDL), toxic injury induced by carbon tetrachloride (CCl4) injections, and fatty liver-associated liver fibrosis induced by feeding methionine- and choline-deficient (MCD) diet using a single intraperitoneal injection of rAAV2/8-ACE2 [87]. The treatment produced a major increase in ACE2 expression and protein activity, which was confined to the liver without affecting other major organs. Unlike inherited disorders, for example, hemophilia B where a relatively low level of transgene expression in the liver may be sufficient for subsequent small increases in FIX levels in the blood [48, 81], the magnitude of the expression of transgene required for therapeutic intervention in non-inherited disease may be substantially higher. This, in turn, may pose a challenge for gene therapy researchers. Interestingly, however in our liver-targeted therapeutic approach with rAAV2/8-ACE2, we found that increased hepatic ACE2 expression reduced hepatic level of profibrotic Ang II by more than 50% compared to those treated with a control vector that carried human serum albumin (rAAV2/8-HSA) [87]. A reduction of Ang II, which was accompanied by increases in hepatic levels of antifibrotic Ang-(1–7) peptide, resulted in a marked reduction in inflammatory cytokine expression, leading to a profound reduction in hepatic fibrosis in all three models (**Figure 2**) [87]. These studies with short-term animal models have been further validated to provide evidence that in long-term animal models of biliary fibrosis and fatty liver disease, which produce hepatic lesions more comparable to those seen in patients with such diseases, a single intraperitoneal injection of rAAV2/8-ACE2 caused a profound reduction in hepatic fibrosis (**Figure 3**). In marked contrast to other studies using

AAV vectors [93], we found that rAAV2/8-ACE2 reduced serum alanine transaminase (ALT) levels in diseased animals compared to those that received the control vector (rAAV2/8-HSA), suggesting that the vector itself is safe in the liver. Moreover, rAAV2/8-HSA (up to 10 days) or rAAV2/8-ACE2 (up to 24 weeks) vector injected into healthy mice produced no change in plasma ALT level, confirming that the vector itself is unlikely to cause liver injury [6, 87]. The schematic representation of molecular mechanism associated with ACE2 gene therapy using

**Figure 3.** rAAV2/8-ACE2 therapy in Mdr2-KO mice with hepatic fibrosis. rAAV2/8-ACE2 gene therapy has markedly increased the ACE2 gene expression in Mdr2-KO mice, whereas liver fibrosis was significantly reduced by the therapy

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

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

Liver-targeted gene delivery using rAAV2/8 vector has shown to be therapeutically promising in adult liver, but their effects have not been extensively investigated in the immature liver. Although rAAV2/8 transduces neonatal mouse liver with high efficiency, the vector is not persistent in the liver and declines rapidly with liver growth [127]. Therefore, the successful use of rAAV2/8-mediated therapy to treat liver disease in early childhood may require readministration [128]. In line with this, another study demonstrated that the treatment of ornithine transcarbamylase (OTC)-deficient neonatal mice with AAV2/8-OTC therapy failed to protect mice from hyperammonemia in adulthood [129]. Thus, producing stable transduction in the developing liver remains one of the biggest challenges for liver-specific rAAV2/8 gene therapy, and readministration of vectors may be necessary to maintain therapeutic effi-

Although the AAV vectors employed for preclinical studies may be effective in human liver, it is important to select an AAV vector specific for human hepatocytes with enhanced transduction efficiency [6, 55]. Recently, two groups have proposed using humanized mice such as the immunosuppressed FRG (Fah−/−/Rag2−/−/Il2rg−/−) mouse model to identify the best rAAV serotype for liver-directed gene therapy [55, 130]. The studies in humanized

rAAV2/8 vector in hepatic fibrosis is shown in **Figure 4**.

in ACE2-treated mice compared to the control vector-injected Mdr2-KO mice.

cacy in adulthood after early neonatal treatment.

**Figure 2.** Hepatic ACE2 gene expression and fibrosis in three short-term models of liver fibrosis with rAAV2/8-ACE2 therapy. ACE2 gene expression (A–C) was significantly increased (p < 0.0001) in ACE2-treated diseased mice compared to control vector (rAAV2/8-HSA) injected diseased mice of BDL, CCl4, and MCD. As a result, rAAV2/8-ACE2 gene therapy has markedly reduced the liver fibrosis in each mouse model (BDL, CCl4, and MCD).

**Figure 3.** rAAV2/8-ACE2 therapy in Mdr2-KO mice with hepatic fibrosis. rAAV2/8-ACE2 gene therapy has markedly increased the ACE2 gene expression in Mdr2-KO mice, whereas liver fibrosis was significantly reduced by the therapy in ACE2-treated mice compared to the control vector-injected Mdr2-KO mice.

AAV vectors [93], we found that rAAV2/8-ACE2 reduced serum alanine transaminase (ALT) levels in diseased animals compared to those that received the control vector (rAAV2/8-HSA), suggesting that the vector itself is safe in the liver. Moreover, rAAV2/8-HSA (up to 10 days) or rAAV2/8-ACE2 (up to 24 weeks) vector injected into healthy mice produced no change in plasma ALT level, confirming that the vector itself is unlikely to cause liver injury [6, 87]. The schematic representation of molecular mechanism associated with ACE2 gene therapy using rAAV2/8 vector in hepatic fibrosis is shown in **Figure 4**.

Liver-targeted gene delivery using rAAV2/8 vector has shown to be therapeutically promising in adult liver, but their effects have not been extensively investigated in the immature liver. Although rAAV2/8 transduces neonatal mouse liver with high efficiency, the vector is not persistent in the liver and declines rapidly with liver growth [127]. Therefore, the successful use of rAAV2/8-mediated therapy to treat liver disease in early childhood may require readministration [128]. In line with this, another study demonstrated that the treatment of ornithine transcarbamylase (OTC)-deficient neonatal mice with AAV2/8-OTC therapy failed to protect mice from hyperammonemia in adulthood [129]. Thus, producing stable transduction in the developing liver remains one of the biggest challenges for liver-specific rAAV2/8 gene therapy, and readministration of vectors may be necessary to maintain therapeutic efficacy in adulthood after early neonatal treatment.

Although the AAV vectors employed for preclinical studies may be effective in human liver, it is important to select an AAV vector specific for human hepatocytes with enhanced transduction efficiency [6, 55]. Recently, two groups have proposed using humanized mice such as the immunosuppressed FRG (Fah−/−/Rag2−/−/Il2rg−/−) mouse model to identify the best rAAV serotype for liver-directed gene therapy [55, 130]. The studies in humanized

**Figure 2.** Hepatic ACE2 gene expression and fibrosis in three short-term models of liver fibrosis with rAAV2/8-ACE2 therapy. ACE2 gene expression (A–C) was significantly increased (p < 0.0001) in ACE2-treated diseased mice compared to control vector (rAAV2/8-HSA) injected diseased mice of BDL, CCl4, and MCD. As a result, rAAV2/8-ACE2 gene

therapy has markedly reduced the liver fibrosis in each mouse model (BDL, CCl4, and MCD).

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

mouse model repopulated with over 25% human hepatocytes allowed the researchers to identify human liver-specific AAV vectors such as LK-03 derived from capsid DNAshuffled AAV library. This library was generated using 10 AAV capsid genes. LK-03, which is composed of five different parental AAV capsids, was able to transduce human primary hepatocytes at higher efficiency *in vitro* and in a hepatocellular carcinoma xenograft model *in vivo* when compared to AAV serotype 8 [55]. Wang and colleagues also reported a higher liver transduction level in FRG mice using capsid of AAVrh10, a clade E AAV derived from rhesus macaque, and AAV3B and have shown that AAV-LK-03 vectors may be superior to either AAV3B or AAV8 [131]. It is expected that researchers will increasingly use humanized animal models for diseases other than liver disease, which will allow them to identify novel variants of engineered AAV vectors, transduction efficiency, and immune reactions specific to the human tissue under investigation. Moreover, it has been reported that AAV3B-eGFP vector, which was able to cause liver-specific robust GFP expression in the livers of non-human primates, is significantly better than AAV8 with no apparent

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

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

Much of preclinical studies which employed a diverse range of naturally occurring as well as engineered AAV vectors in the last decade provided ample evidence that therapeutic gene transfer certainly holds a great promise for patients with inherited disorders such as those that developed as a result of blood clotting factor deficiency and mutated retinal genes causing blindness. Moreover, it is now becoming clear that the findings of preclinical studies of noninherited disorders suggest that clinical studies utilizing therapeutic gene transfer is feasible. Currently active clinical trials in patients with inherited disorders using a diverse range of AAV vector types will be expected to provide valuable insights into the safety and efficacy of AAV vectors [133]. Since the FDA as well as the EU has now endorsed human gene therapy, there is every possibility that the volume of gene therapy research employing next-generation AAV vectors for both inherited and non-inherited disorders in both preclinical and clinical settings would be expected to increase in the coming years. Moreover, a rapidly evolving technology of AAV vector engineering and the use of humanized animal models would be a key for rapid translation of preclinical findings to clinical studies. The findings from our ongoing liver fibrosis/cirrhosis work using human liver-specific AAV-LK-03 vector in humanized FRG mice would be expected to provide valuable information before we commence clini-

hepatotoxicity [132].

**13. Conclusions**

**Author details**

cal studies in patients with chronic liver disease.

Indu Rajapaksha, Peter Angus and Chandana Herath\*

The University of Melbourne, Melbourne, Australia

\*Address all correspondence to: cherath@unimelb.edu.au

**Figure 4.** rAAV2/8-ACE2 uptake by hepatocytes and a cascade of events triggered by ACE2 protein in activated hepatic stellate cells (HSCs) during fibrosis. rAAV-ACE2 particles use AAV receptor (AAV-R) on hepatocyte membrane to enter the cytoplasm, followed by translocation into nucleus where uncoating and releasing of single-stranded viral genome occurs. The complementary strand will then be synthesized to transcribe ACE2. Membrane bound ACE2 protein has an exclusive role of cleaving potent profibrotic peptide angiotensin II (Ang II) to antifibrotic peptide angiotensin-1-7 (Ang-(1–7)). While a reduction in local Ang II levels leads to a significant reduction in the activation of its receptor, Ang II type 1 (AT1-R), Ang-(1–7) working through its receptor, Mas (Mas-R), inhibits the AT1-R activated downstream signaling such as PKC- and NADPH-mediated ROS production in activated HSCs. This in turn inhibits the phosphorylation of MAPKs such as ERK1/2, JNK, and p38, leading to a reduction in proinflammatory cytokines such as IL-1, IL-6, IL-8, IFNγ, MCP-1, and TNFα and profibrotic cytokine TGFβ1. A reduction in the activity of TGFβ1 leads to a reduction in phosphorylation of its transcription factors, Smad2/3, resulting in the inhibition of secretion of matrix proteins such as collagens and fibronectins. Thus, rAAV-ACE2 helps improving hepatic fibrosis and thus, intrahepatic vascular tone, leading to an improvement in portal hypertension. PKC, protein kinase C; NADPH oxidase, nicotinamide adenine dinucleotide phosphate oxidase; IL, interleukin; IFNγ, interferon γ; MCP-1, monocyte chemotactic protein 1; TNFα, tumor necrosis factor α; TGFβ1, transforming growth factor-β1; ERK1/2, extracellular regulated kinase1/2; JNK, C-Jun N-terminal kinase.

mouse model repopulated with over 25% human hepatocytes allowed the researchers to identify human liver-specific AAV vectors such as LK-03 derived from capsid DNAshuffled AAV library. This library was generated using 10 AAV capsid genes. LK-03, which is composed of five different parental AAV capsids, was able to transduce human primary hepatocytes at higher efficiency *in vitro* and in a hepatocellular carcinoma xenograft model *in vivo* when compared to AAV serotype 8 [55]. Wang and colleagues also reported a higher liver transduction level in FRG mice using capsid of AAVrh10, a clade E AAV derived from rhesus macaque, and AAV3B and have shown that AAV-LK-03 vectors may be superior to either AAV3B or AAV8 [131]. It is expected that researchers will increasingly use humanized animal models for diseases other than liver disease, which will allow them to identify novel variants of engineered AAV vectors, transduction efficiency, and immune reactions specific to the human tissue under investigation. Moreover, it has been reported that AAV3B-eGFP vector, which was able to cause liver-specific robust GFP expression in the livers of non-human primates, is significantly better than AAV8 with no apparent hepatotoxicity [132].
