*2.1. Clinical Applications*

Gene addition therapies driven by Adeno Associated Viral (AAV) vectors for recessive monogenic diseases have been developed and, upon showing safety and efficacy, have received market approval, albeit in a limited number. Presently, three AAV-vector based gene therapeutics are available as Glybera, treating Lipoprotein Lipase (LPL)-deficiency, Luxturna [28], treating Retinal Pigment Epithelial (RPE) 65-related retinal dystrophy, and Zolgensma, treating Spinal Muscular Atrophy (SMA) [28–30]. The promising safety and efficacy data obtained in small clinical AAV gene therapy studies resulted in their application for registration to obtain market approval. For instance this approach as a treatment for Choroidenemia [31], aromatic L-amino acid decarboxylase deficiency [32], Pompe's Disease [33,34], Duchenne Muscular Dystrophy (ongoing: NCT03375164), Becker's Muscular Dystrophy [35], Limb-Girdle Muscular Dystrophy [36], X-linked myotubular myopathy (ongoing: NCT03199469), haemophilia A [37] and haemophilia B [38,39] all show efficacy. However, some safety issues have occurred, especially in clinical trials for diseases requiring systemic high vector doses. Liver toxicity has been reported for AAV doses above 1 × 10−<sup>14</sup> vg/kg, indicating that this will be a problem for some applications [40]. Gene therapies for ocular diseases have relatively few hurdles to surmount because the eye is easily accessible, highly compartmentalized, immune-privileged [41], and less invasive as the injection occurs non-systemically. SMA treatment with Zolgensma, targeting the motor neurons, urges the use of intrathecal injections and a high vector doses, causing transient liver inflammation [42,43]. Recently, in a trial targeting X-linked myotubular myopathy, there have been two tragic deaths of pediatric patients in the cohort receiving the highest vector dose [44]. Both patients died due to progressive liver dysfunction and subsequently fatal sepsis upon receiving 3 × 10−<sup>14</sup> vg/kg. Patients treated with a lower vector dose, 1 × 10−<sup>14</sup> vg/kg, did not experience any severe adverse effects. The liver tropism of AAV vectors, resulting in toxic vector levels in the hepatocytes complicates gene therapy of disorders requiring a high vector dose in the systemic circulation, like for instance all muscular disorders. Within the scope of this review, looking at gene therapy for PFIC, AAV-mediated gene transfer directed to the liver is especially relevant. When treating inherited liver disorders, the AAV tropism for the liver is a major advantage since lower vector doses are needed for therapeutic efficacy. A number of clinical trials to treat Factor VIII or Factor IX deficiency in hemophilia A or B patients have our equitable interest.

The three year follow-up of a dose escalation study to treat haemophilia A, performed by Pasi and colleagues [37], showed long-term efficacy. All 15 adult patients received doses of 6 × 10−<sup>12</sup> vg/kg up to 6 × 10−<sup>13</sup> vg/kg of AAV5-h*FVIII* intravenously. No hepatotoxicity was observed, although in some cases elevated Aspartate Transaminase (AST) levels occurred and were successfully treated with glucocorticoids. Factor VIII plasma activity was restored leading to strong decreases in bleeding incidences, and recombinant Factor VIII use. Although after three years, factor VIII plasma levels were still therapeutic, a gradual decrease over time was seen. A longer follow up is needed to learn if factor VIII levels will remain therapeutic or if re-treatment will be needed. Dose escalation studies to treat Factor IX deficiency also proved to be safe and effective since levels up to 40–50% of normal Factor IX activity were reached [39]. Long-term follow up of an older study for Factor IX deficiency showed long-term efficacy [38]. In these patients the AAV8 vector was injected over nine years ago and they still have clinically relevant Factor IX production. To conclude, AAV-mediated liver-directed gene therapy as a one-time treatment proved to be safe and to provide long-term correction in adults, indicating that this treatment strategy is feasible for inherited severe liver disorders.

AAV vectors do not integrate actively in the host genome but persist in the nucleus in episomal form [45,46]. An important advantage of this is the lack of genotoxicity which can for instance result in tumour formation, as seen with integrating retroviral vectors [47,48]. During cell division, these episomes are not copied and not distributed to the daughter cells. This renders AAV vectors less suitable for treatment of liver disorders early after birth. The growth of the liver results in loss of the initial efficacy [49,50]. Hepatocyte proliferation is also induced upon liver damage for instance after a partial hepatectomy [51]. This complicates the application of AAV-mediated gene therapy for disorders resulting in hepatocyte damage, such as Fumarylacetoacetate hydrolase (FAH) deficiency and PFIC. Ongoing safety and efficacy studies for AAV gene therapy are performed in adults targeting diseases that do not cause liver damage. Recent pre-clinical studies do indicate that AAVmediated gene therapy for PFIC may be feasible.
