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

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].

**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,

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

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

and integrity of the packaged AAV genome [87].

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

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 gene therapy for hemophilia B.

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

enzyme levels, they were recovered after a short-term prednisone treatment. Of all participants, only one patient had been treated with FIX concentrates who was diagnosed with an advanced arthropathy at baseline. However, the use of FIX concentrate was reduced to 91% comparing to the status before vector infusion. Additional clinical trials are underway with AAV2/8 hFIX (NCT00979238) and FIX-Padua (NCT01687608), which will provide more information on safety and efficacy of the therapy [103]. Overall, the results from these studies suggest that gene therapy has the potential to significantly improve disease phenotype in hemophilia B patients. It is of significance that for the first time, the US Food and Drug Administration (FDA) has approved a pioneering gene therapy protocol using an AAV vector for a rare form of childhood blindness in 2017 as the first such treatment cleared in the United States for an inherited disease. The disease known as Leber congenital amaurosis (LCA) develops due to mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene, causing a severe form of inherited retinal blindness in infants and children. Several independent studies [104–106] using rAAV2/2 expressing RPE65 complementary DNA (cDNA) have provided preliminary evidence of shortterm safety and efficacy in this disorder. Further studies by Cideciyan and colleagues showed a significant efficacy of human retinal gene transfer with rAAV2-RPE65 vector with transgene expression for up to 1 year post treatment [107]. Also, they have proven the treatment as a safe therapy by evaluating the safety parameters obtained through regular standard eye examinations, physical examinations, routine hematology, serum chemistries, coagulation parameters, and urinalysis. This particular FDA-approved gene therapy (LUXTURNA) (voretigene neparvovec-rzyl) is to be used in patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy. This approval is considered as a milestone of AAV vector-associated gene therapy research and further encourages researchers to develop successful vectors to deliver

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

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

therapeutic genes for number of diseases where there is no effective medical treatment.

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

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

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

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 no recurrent elevation of serum ALT in patients was observed.

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 enzyme levels, they were recovered after a short-term prednisone treatment. Of all participants, only one patient had been treated with FIX concentrates who was diagnosed with an advanced arthropathy at baseline. However, the use of FIX concentrate was reduced to 91% comparing to the status before vector infusion. Additional clinical trials are underway with AAV2/8 hFIX (NCT00979238) and FIX-Padua (NCT01687608), which will provide more information on safety and efficacy of the therapy [103]. Overall, the results from these studies suggest that gene therapy has the potential to significantly improve disease phenotype in hemophilia B patients.

It is of significance that for the first time, the US Food and Drug Administration (FDA) has approved a pioneering gene therapy protocol using an AAV vector for a rare form of childhood blindness in 2017 as the first such treatment cleared in the United States for an inherited disease. The disease known as Leber congenital amaurosis (LCA) develops due to mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene, causing a severe form of inherited retinal blindness in infants and children. Several independent studies [104–106] using rAAV2/2 expressing RPE65 complementary DNA (cDNA) have provided preliminary evidence of shortterm safety and efficacy in this disorder. Further studies by Cideciyan and colleagues showed a significant efficacy of human retinal gene transfer with rAAV2-RPE65 vector with transgene expression for up to 1 year post treatment [107]. Also, they have proven the treatment as a safe therapy by evaluating the safety parameters obtained through regular standard eye examinations, physical examinations, routine hematology, serum chemistries, coagulation parameters, and urinalysis. This particular FDA-approved gene therapy (LUXTURNA) (voretigene neparvovec-rzyl) is to be used in patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy. This approval is considered as a milestone of AAV vector-associated gene therapy research and further encourages researchers to develop successful vectors to deliver therapeutic genes for number of diseases where there is no effective medical treatment.
