Advanced Therapies for Inborn Errors of Metabolism: Development of innovative approaches to treat Pompe Disease and Crigler-Najjar Syndrome

Inborn errors of metabolism (IEMs) are a large group of rare, inherited, often fatal disorders that affect metabolic pathways, due to deficient enzymes, cofactors, or transporters. To date, while a better understanding of these complex disorders have led to significant progress in the development of many new treatments, most IEMs remain without curative treatment. Because of their severity and the progresses made in early diagnosis, IEMs represent attractive targets for gene therapy. In the recent years, adeno-associated virus (AAV) vectors have emerged as a promising approach for treating this heterogeneous group of disorders, with a growing number of ongoing clinical trials. The overall goal of the works carried out during this thesis was to develop and support clinical application of AAV-based gene therapies for rare IEMs. In particular, we focused our efforts in the design, optimization and preclinical development of innovative approaches to treat Pompe disease (PD) and Crigler-Najjar (CN) syndrome. PD is a lysosomal storage disorder and neuromuscular disease, with systemic, multi-organ manifestations. In PD patients, glycogen is no longer broken down effectively into glucose, due to a deficiency of the lysosomal enzyme acid α-glucosidase (GAA). So far, no curative treatment is available for PD. The complexity and diversity of this disease, has prompted us to optimize AAV gene therapy-based approaches. We have worked to find solutions to improve the muscular and neurological symptoms of the disease, through substrate reduction therapy (SRT) or the amelioration of gene transfer efficacy in target tissues. Notably, we demonstrated the feasibility of a miRNA-based and genome editing-based SRT using an AAV-mediated gene therapy approach, evidencing efficacy to prevent glycogen build-up in Pompe mice treated as neonates. We also tested highly optimized myospecific AAV vectors that represent promising tools to improve muscle gene therapy delivery. Finally, we attempted to enhance the GAA delivery in muscle and CNS after AAV-mediated liver gene transfer, by using pharmacological chaperones therapy (PCT). Together, this thesis shows the results of different approaches that might increase the safety and efficacy of AAV gene therapy for PD. CN is a life-threatening, incurable liver disease that affects about one in a million individuals at birth. It is characterized by a deficiency of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme that leads to the accumulation of high levels of bilirubin in all body tissues. If not treated, brain accumulation of bilirubin leads to neurological damage and death. The current management of CN is based on the ability of blue light to degrade bilirubin. Patients are exposed to phototherapy (PT) for 10-12 hours per day. However, despite the relative efficacy of this treatment to maintain bilirubin under a toxicity level, PT has several shortcomings, leaving liver transplantation the only curative option for severely affected individuals. Gene therapy represents an alternative curative option to correct the genetic defect, restore the enzyme expression and the consequent bilirubin conjugation. We developed an investigational liver-directed gene therapy based on an optimized AAV8 vector injected intravenously to deliver a corrected copy of the UGT1A1 gene to hepatocytes. Our work demonstrated the safety and efficacy of this approach in different relevant animal models, and supported the clinical translation of AAV8-UGT1A1 gene therapy for the treatment of CN syndrome.