409. Complete Correction of Enzymatic Deficiency and Lysosomal Storage throughout the Brain in a Mouse Model of GM1-gangliosidosis after Neonatal Intracerebroventricular Injection of an AAV1 Vector Encoding Lysosomal Acid Beta- galactosidase
Authors: Marike L. D. Broekman, Rena Baek, Laryssa A. Comer, Thomas N. Seyfried, Miguel Sena-Esteves
Journal: Molecular Therapy (2006)

GM1 gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by autosomal recessive deficiency of lysosomal acid |[beta]|-galactosidase (|[beta]|gal). This disease is characterized by accumulation of GM1-ganglioside and its asialo-form (GA1) in the CNS. In this study we examined the effect of neonatal intracerebroventricular injection of an adeno-associated virus (AAV) vector (serotype 1) encoding mouse |[beta]|gal under control of a CBA promoter (AAV1-CBA-|[beta]|gal), on bgal enzyme activity and brain GSL content in adult GM1 gangliosidosis (|[beta]|gal-/-) mice. A total of 4 |[mu]|l of AAV1-CBA-|[beta]|gal vector with a titer of 4.3x1013 gc/mL was injected into the lateral ventricles (2 |[mu]|l each) of P0 |[beta]|gal-/- mice. At 3 months we analyzed one brain hemisphere for enzyme distribution by X-gal staining at pH 5.0. |[beta]|gal was expressed throughout the brain at higher levels than those found in wild type controls or untreated |[beta]|gal-/- mice. The highest levels of enzymatic activity were associated with allocortical areas such as the hippocampal formation and olfactory bulb, and periallocortical areas. Five regions in the other hemisphere were analyzed for enzymatic activity and GSL content. AAV1-treated |[beta]|gal-/- mice presented enzymatic activities between 7- and 65-fold higher (depending on the region) than in wild type controls. Total brain ganglioside and GM1 content (|[mu]|g of sialic acid/100 mg dry weight) was reduced from 824 |[plusmn]| 65 |[mu]|g and 476 |[plusmn]| 42 |[mu]|g in non-treated |[beta]|gal -/- mice to 444 |[plusmn]| 21 |[mu]|g and 46.9 |[plusmn]| 2.1 |[mu]|g in AAV1-treated |[beta]|gal -/- mice, respectively. These GSL values in treated mice were statistically identical to those measured in wild type controls (412 |[plusmn]| 17 |[mu]|g and 53.2 |[plusmn]| 1.1 |[mu]|g, respectively). Furthermore, GA1 storage was completely eliminated. Thus GSL levels in the brains of AAV-treated |[beta]|gal-/- mice were restored to normal levels. Analysis of GM1-ganglioside storage in the brain by staining with subunit B of cholera toxin (CTX-B) showed that AAV-treated |[beta]|gal-/- mice were identical to wild type controls. Interestingly, fiber tracts in untreated |[beta]|gal-/- mice stained weakly with CTX-B or not at all. This may be the mouse equivalent of delayed myelination reported in type I GM1-gangliosidosis in humans and dogs. Filipin staining of brains for unesterified cholesterol, which accumulates in the brain in glycosphingolipidoses, showed that AAV1-treated |[beta]|gal-/- mice were indistinguishable from wild type mice, while untreated |[beta]|gal-/- mice displayed strong storage throughout the brain. Altogether the biochemical and histological evidence in this study show that brain metabolism in AAV-treated |[beta]|gal-/- mice was normal. Thus we conclude that neonatal AAV- mediated gene delivery of lysosomal acid |[beta]|-galactosidase to the brain is an effective approach for treatment of GM1 gangliosidosis.