ABSTRACT Glycogen storage disease type III (GSD III) is an autosomal recessive inherited disorder caused by deficiency of glycogen debranching enzyme (GDE) that leads to excessive accumulation of abnormal glycogen (limit dextrin) in muscle and liver tissues. The majority of patients (~85%) have both muscle and liver involvement (GSD IIIa) while others have disease limited to the liver (GSD IIIb). In absence of an effective therapy, patients with GSD III are experiencing progressive liver failure and muscle damage accompanied by increased morbidity and mortality. Adeno-associated virus (AAV) mediated gene therapy has shown promise for treating inherited muscle and liver disorders with successful translation to clinical trials. However, this approach has not been advanced for GSD III because AAV is not capable of delivering the large (4.6 kb) human GDE cDNA, due to its small packaging capacity. We have developed an innovative gene therapy approach with AAV9 in a mouse model of GSD IIIa with two key components incorporated 1) a small bacterial GDE analog to overcome the limitation of small AAV carrying capacity; and 2) a novel immunotolerizing dual promoter to prevent cytotoxic T lymphocyte (CTL) response to the bacterial enzyme and enable long-term Pullulanase expression in all affected tissues. The overall objective of this project is to identify a path forward for clinical translation of this promising therapy. It is commonly known that the therapeutic outcomes of AAV vectors in mouse models does not always translate into human. Current AAV serotypes, especially AAV9, transduce muscle and liver in mice with high efficiency; however, high doses of AAV9 required to transduce skeletal muscles in human patients can led to adverse hepatotoxicity or even liver failure. In this proposal, we aim to identify a lead therapeutic candidate AAV vector in GSD IIIa mice using high potency cross-species compatible AAV capsids (ccAAVs) containing a de- immunized transgene expression cassette to minimize gene therapy related immune responses and reduce the effective vector dose (Aim 1a). We will validate the lead AAV vector in GSD IIIa patient muscle cells and human liver chimeric mice to increase its clinical translatability (Aim 1b). We will then examine the long-term efficacy of the lead AAV vector in GSD IIIa mice (Aim 2), and test its safety and efficacy in GSD IIIa dogs (Aim 3). Data generated from the proposed studies will lay the foundation for translating this innovative gene therapy to patients with GSD III. The concept of using a bacterial enzyme to treat human diseases through gene therapy may open up new alternatives for therapeutic development for metabolic disorders caused by defects in large genes. The immunotolerizing dual promoter technology can also be broadly used for treating other conditions that affect multiple tissues with gene therapy.

PROJECTIVE NARRATIVE Data generated from the proposed studies will lay the foundation for translating an innovative gene therapy approach to patients with GSD III. The technologies to be utilized in this proposal can be broadly used for gene therapy development for other inherited disorders.