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GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline β-galactosidase (β-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where β-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and β-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

The G(M2) activator protein is required for successful degradation of G(M2) ganglioside by the A isozyme of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52). Deficiency of the G(M2) activator protein leads to a relentlessly progressive accumulation of G(M2) ganglioside in neuronal lysosomes and subsequent fatal deterioration of central nervous system function. G(M2) activator deficiency has been described in humans, dogs and mice. This manuscript reports the discovery and characterization of a feline model of G(M2) activator deficiency that exhibits many disease traits typical of the disorder in other species. Cats deficient in the G(M2) activator protein develop clinical signs at approximately 14 months of age, including motor incoordination and exaggerated startle response to sharp sounds. Affected cats exhibit central nervous system abnormalities such as swollen neurons, membranous cytoplasmic bodies, increased sialic acid content and elevated levels of G(M2) ganglioside. As is typical of G(M2) activator deficiency, hexosaminidase A activity in tissue homogenates appears normal when assayed with a commonly used synthetic substrate. When the G(M2) activator cDNA was sequenced from normal and affected cats, a deletion of 4 base pairs was identified as the causative mutation, resulting in alteration of 21 amino acids at the C terminus of the G(M2) activator protein.

Feline ganglioside storage diseases are authentic models of the human conditions and are valuable for development of gene therapy strategies for human patients. Deficiency of the lysosomal enzymes |[beta]|-galactosidase or |[beta]|-N-acetylhexosaminidase causes continuous cumulation of GM1 or GM2 ganglioside, respectively, resulting in disease manifestations nd death. There is no reliable treatment for human gangliosidoses, although knockout mouse models have been used to develop promising experimental treatments including gene therapy with adeno-associated virus vectors. Intracranial injection of GM1 knockoutenetic background (FVB-Twi), that we generated based on previous indications on the effects of the genetic background in modulating GLD severity (Tominaga K. et al., J. Neurosci. Res. 2004; Biswas S. et al., Neurobiol. of Disease 2002); ii) a mouse with low GALC activity, recently generated in D. Wenger's laboratory by introducing in the murine GALC gene a polymorphism that was found in individuals having some not otherwise explainable neurological abnormalities in the presence of 10|[ndash]|20% residual GALC activity, the trs mouse (Luzi P. et al., Mol. Genetics and Metab. 2001). These three models showed some substantial differences in their phenotype and mice has achieved supranormal levels of |[beta]|-galactosidase throughout the brain, with lower levels of |[beta]|-galactosidase activity observed at distant sites including retina and spinal cord (M. Sena-Esteves, unpublished data). Similarly, intracranial injection of GM2 knockout mice has produced widespread expression of hexosaminidase activity, substantially decreased storage of GM2 ganglioside and life spans significantly increased to greater than three times that of untreated GM2 mice (see abstract by Cachon-Gonzalez at this meeting). To investigate the potential for adeno-associated virus treatment of human patients, well-characterized feline models of gangliosidoses were treated with vectors proven to be successful in mice. After intracranial injections of vector through a single needle track, enzymatic activity was detected throughout the injected hemisphere, with high levels at the injection site and gradually decreasing but substantial enzymatic activity at least 1 cm cranial and caudal to the point of injection. Enzymatic activity also was detected in the cerebral cortex of the contralateral hemisphere. In addition, staining for storage material with the periodic acid-Schiff (PAS) reagent indicated that decreased ganglioside content coincided with increased levels of enzymatic activity. Detailed results of these studies will be presented.