**8. Clinical trials for hemophilia B treatment**

Furthermore, visual function was improved in the 15 patients with a variable degree [38]. However, between 9 and 12 months of gene therapy administration, four of the fifteen patients

Figure <sup>9</sup>. Diseases currently being tested in clinical trials with different rAAV **Figure 9.** Diseases currently being tested in clinical trials with different rAAV serotypes.

Furthermore, the number of studies that were in phase III was reduced, as the therapies started to reach the market. For instance, in October 2012,

Glybera became the first rAAV gene therapy to obtain marketing

Since their discovery in the 1960s as small DNA viruses contaminating

have consistently confirmed that rAAV vector delivery is safe, well tolerated by humans and efficient in transferring the therapeutic gene. Figure 8B summarizes the spectrum of diseases that have been tested with rAAV gene therapy in 2010 and 2015. The statistics show that neurological and ocular diseases are gaining more interest, probably because they both

constitute immunological privileged tissues. Figure 9 summarizes the diseases that are being treated with AAV technology, according to the

cultures of simian and human adenoviruses [2, 34], AAV vectors have been tested in more than a hundred clinical trials. Completed and ongoing trials

authorization from the European Commission.

The company Sparks, which is sponsoring the studies in the US, is testing the technology developed at the Children's Hospital of Philadelphia in a phase 3 trial and expects to announce their results in 2015. If the results are promising, it could be the next rAAV gene therapy

Among the brain diseases, Parkinson's treatment was attempted with rAAV gene therapy delivering different transgenes. Administration of glutamic acid decarboxylase (GAD) via rAAV2 produced modest efficacy improvements. Patients were injected with rAAV2 coding for GAD65 and GAD67 in the center of the subthalamic nucleus [40]. Six months following the injection, the unified Parkinson's disease rating scale decreased by 8.1 points, compared with a reduction of 4.7 points that the sham operation group evidenced. Six months later, clinical improvements were still being noticed. However, the results were modest and the protocol had some deviations. For instance, patients who showed no benefit on the primary endpoint were eliminated from the statistical analysis, arguing that the injections were off-target [41]. Administration of aromatic L-amino acid decarboxylase (AADC) gene was tested on a phase I trial that consisted of the treatment of 15 patients with moderate disease [42, 43]. The trial, sponsored by Genzyme, observed only a modest efficacy, results that were confirmed by a second study performed in Japan [44]. Similarly, phase I and II trials with the rAAV2-neurturin (CERE-120) vector from Ceregene failed to show statistically significant improvement in the rAAV-treated group compared with the group that received the sham surgery [45, 46]. As a conclusion of all these different trials, the technology is safe and is promising. However,

experienced new pseudo-foveas in the retinal regions, for up to six years [39].

product to be launched in the market.

serotype.

132 Gene Therapy - Principles and Challenges

Hemophilia B is an X-linked recessive disorder, which originated from mutations within the gene that encodes the coagulation factor IX. Therefore, patients whose functional FIX levels are 1% of normal levels will bleed into the joint and muscle tissues. Bleeding in the brain could result in fatal death. If FIX levels are between 1% and 5%, the individual will experience a reduced number of bleeding incidents and a moderate phenotype of the disease. Any FIX levels above 5% will allow the person to have a normal life [53]. The only available treatment is protein replacement therapy, which requires regular intravenous injections and is expensive. Therefore, novel and permanent therapies/treatments are urgent. rAAV gene therapy cur‐ rently constitutes a promising approach for the treatment of several diseases, including hemophilia B.

Based on animal studies that were described in the previous section, four clinical trials have been initiated. The first study administered rAAV carrying FIX gene into three patients by intramuscular injection. Despite the presence of preexistent high titer of neutralizing antibod‐ ies against capsid rAAV, strong transgene expression was observed in the muscle, even after 10 months of injection. However, levels of factor IX in circulation were less than 1–2% in most cases, even at the highest tested dose. Toxicity was not observed [54, 55].

The second trial conducted by the University of Pennsylvania infused the virus through the hepatic artery into seven patients. The rational in this protocol considered that FIX is a secreted protein and once it is produced and reaches the bloodstream; it can be distributed throughout the body.

Even though levels of FIX resulted higher than 5% after injecting an intermediate or high viral dose, the therapeutic effect was only transient (up to eight weeks), due to the development of a strong cytotoxic T response, which destroyed the transfected hepatocytes and thus hampered the production of FIX [56].

The third trial was designed in order to increase FIX expression production as well as to circumvent the possibility of a humoral response that could interfere with the success of the therapy [57]. To reach the first goal, they developed a codon optimized FIX gene that also delivered the gene in the context of self-complementary rAAV, which provides substantially higher levels of transgene expression rather than delivering the WT gene with single-stranded rAAV. In order to reduce antibody neutralization, the viral genome cassette was packed in rAAV8 capsid, as it has lower seroprevalence in humans and a high tropism for the liver. The virus was administered directly in the peripheral vein in six patients, and all of them developed 1–6% levels of factor IX expression in the first four months as well as for at least three years. There was no modification on the levels of neutralizing antibodies. However, transient elevations in serum liver enzymes, possibly as a result of a cellular immune response to the rAAV8 capsid, were observed in the three patients who received the high viral dose.

Recently, Baxter's laboratories launched a clinical trial to test the safety and efficacy of a selfcomplimentary rAAV8 vector carrying a mutant FIX sequence (BAX 335), created and preclinically tested at the UNC gene therapy center [58]. The FIX sequence had a single amino acid change at position 338, which substantially increased the levels of circulating FIX protein. A more effective rAAV delivery vehicle allowed the administration of lower viral doses with the same efficacy as previous tested virus, but without the associated toxicity. In this trial, sixteen adult patients were treated in different centers around the US. Updates on the trial were presented at the 8th Annual Congress of the European Association for Haemophilia and Allied Disorders this year in Finland. At this time, patients who received the highest dose did not develop inhibitory antibodies, reached FIX protein activity of 10% or more and did not manifest bleeding events. However, one of the patients experienced elevated levels of liver enzymes probably due to an immune response.

Nowadays, investigators are still developing better strategies to overcome the immunological response; currently, there are six trials evaluating safety and efficacy, including BAX 335.
