1124. Correction of Glycogen Storage Disease Type II by an AAV Vector Encoding Highly-Secreted, Chimeric Acid α-glucosidase

Abstract

Top of pageAbstract GSD-II (Pompe disease; MIM 232300) is an autosomal recessive disorder caused by the deficiency in the activity of the lysosomal enzyme acid |[alpha]|-glucosidase (GAA; acid maltase; EC 3.2.1.20). The disease is characterized by the massive accumulation of glycogen in lysosomes and in the cytoplasm of striated muscle, with an accompanying disruption of cellular functions. The efficacy of therapy with lysosomal enzymes, such as GAA, could be markedly improved if secretion of these proteins from producer cells were increased. Secreted proteins are directed extracellularly by the presence of an N-terminal signal peptide. We have investigated the hypothesis that a chimeric lysosomal enzyme containing an alternative signal peptide will achieve much higher secretion of the recombinant lysosomal enzyme from transduced cells. The coding sequence of human GAA was modified by substituting alternative signal peptides from highly secreted proteins for the native GAA signal peptide (denoted chimeric GAA). The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold, compared to the secretion of unmodified GAA. The efficacy of this strategy for gene therapy in Pompe disease was analyzed in GAA-knockout (GAA-KO) mice. An adeno-associated virus (AAV) vector containing a liver-specific promoter to drive the expression of GAA was constructed (AAV-LSPSPhGAApA) and pseudotyped as AAV8. The long-term efficacy of SP-hGAA was evaluated by the quantitation of GAA activity and glycogen content following AAV-LSPSPhGAApA administration. A dose-response analysis revealed sustained, high GAA activity in plasma following administration of at least 3 |[times]| 1010 particles; however, an equivalent number of particles for a vector encoding unmodified GAA did not elevate GAA activity in plasma. Receptor-mediated uptake of secreted GAA can be demonstrated only if plasma levels exceed a therapeutic threshold, and the GAA activity of the heart and diaphragm were significantly elevated 18 weeks following administration of as few as 1 |[times]| 1010 particles of AAV-LSPSPhGAApA. GAA activity in the quadriceps muscle was increased with as few as 3 |[times]| 1010 particles of AAV-LSPSPhGAApA. The lower threshold for significant reduction of heart, diaphragm, and quadriceps glycogen content with AAV-LSPSPhGAApA was 3 |[times]| 1010 particles. Neither fewer particles for that vector, nor an equivalent number of particles of the analogous vector encoding unmodified GAA affected glycogen content in the quadriceps. Thus, an AAV vector encoding chimeric GAA increased the secretion of hGAA from the liver, and reduced the number of vector particles required to achieve a therapeutic effect in GSD-II mice.