862. Improved Efficacy of Adenovirus-Mediated Gene Therapy for GSD-II Disease by Selective Depletion of Kupffer Cells

Abstract

Glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by a massive accumulation of glycogen in multiple muscle groups, and is caused by the deficiency of intralysosomal acid alpha glucosidase (GAA). Two approaches to the treatment of GSD-II are currently being pursued in our research group: enzyme replacement therapy and gene therapy. Relative to gene therapy, our previous work demonstrated that a single intravenous administration of advanced generation, [E1-,pol-] adenoviral (Ad) based vectors encoding human GAA (hGAA) resulted in efficient hepatic transduction and secretion of high levels of hGAA, consequently, the correction of all affected muscles in a GAA-KO mouse model as well a quail model of GSD-II occurred. The long-term persistence of the improved vector in liver tissues facilitated significant glycogen reduction in multiple muscles (though, with variable responses in extent and duration among the individual muscle group analyzed) for at least 6 months, despite the fact that hGAA is perceived as a foreign antigen in these animal models. Unfortunately, our most recent work suggests that very high doses of Ad vectors must be intravenously injected to facilitate the large amounts of hepatic hGAA secretion required to correct both cardiac and skeletal muscles in these animal models. To determine if this problem can be overcome, we have begun attempts to pre-emptively suppress the innate immune systems of immunocompetent GAA-KO mice, prior to injection with our viral vectors. Macrophages, in particular hepatic Kupffer cells, play a central role in sequestering Ad vectors, as well in triggering early innate immune responses, and hence have a profound impact on the adaptive immune responses following adenoviral vector administration. In this study we evaluated the effect of selective and transient depletion of Kupffer cells (via pretreatment of mice with clodronate liposomes-dichloromethylene-bisphosphonate) one day prior to vector injection. We intravenously injected into the clodronate liposome pretreated GAA-KO mice different dosages of the vector, to determine how removal of Kupffer cells impacted upon the known efficacy of this approach. We found that clodronate pretreatment allowed lower doses of the vector to show greater efficacy. For example, the GAA activities in the clodronate-treated mice were significantly elevated both in plasma and in tissues such as liver, heart, diaphram and quadriceps, as compared to the non-Kupffer cell depleted mice injected with identical doses of the [E1-,pol-]AdhGAA vector. As a result, the pathogenic glycogen accumulations in clodronate liposome pretreated GAA-KO mice were significantly reduced at lower doses of the Ad vector (10–100 fold less) as compared to Ad treated GAA-KO mice that had not been pretreated with clodronate containing liposomes. Our data also suggests however, that liver toxicities were also now apparent at lower vector dosages, suggesting that Kupffer cells act to prevent liver damage by sequestering Ad particles. Whether or not other “Ad induced” toxicities are prevented by Kupffer cell depletion are studies currently ongoing in our laboratories.