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Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.15). ASPA acts to hydrolyze N-acetylaspartate (NAA) into L-aspartate and acetate, but the connection between ASPA deficiency and the failure of proper CNS development is unclear. We hypothesize that one function of ASPA is to provide acetate for the increased lipid synthesis that occurs during postnatal CNS myelination. The gene encoding ASPA has been inactivated in the mouse model of CD, and here we show significant decreases in the synthesis of six classes of myelin-associated lipids, as well as reduced acetate levels, in the brains of these mice at the time of peak postnatal CNS myelination. Analysis of the lipid content of white matter from a human CD patient showed decreased cerebroside and sulfatide relative to normal white matter. These results demonstrate that myelin lipid synthesis is significantly compromised in CD and provide direct evidence that defective myelin synthesis, resulting from a deficiency of NAA-derived acetate, is involved in the pathogenesis of CD.

Canavan’s disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤1 month) AspA−/− mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.

Background Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N‐acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock‐out mouse for Canavan disease that can be used for gene transfer. Methods Genomic library λ knock‐out shuttle (λKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. Results Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. Conclusion The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases. Copyright © 2000 John Wiley & Sons, Ltd.

Canavan's disease (CD) is a fatal, hereditary disorder of CNS development that has been linked to mutations in the gene for the enzyme aspartoacylase (ASPA) (EC 3.5.1.15). ASPA acts to hydrolyze N-acetylaspartate (NAA) into l-aspartate and acetate, but the connection between ASPA deficiency and the failure of proper CNS development is unclear. We hypothesize that one function of ASPA is to provide acetate for the increased lipid synthesis that occurs during postnatal CNS myelination. The gene encoding ASPA has been inactivated in the mouse model of CD, and here we show significant decreases in the synthesis of six classes of myelinassociated lipids, as well as reduced acetate levels, in the brains of these mice at the time of peak postnatal CNS myelination. Analysis of the lipid content of white matter from a human CD patient showed decreased cerebroside and sulfatide relative to normal white matter. These results demonstrate that myelin lipid synthesis is significantly compromised in CD and provide direct evidence that defective myelin synthesis, resulting from a deficiency of NAA-derived acetate, is involved in the pathogenesis of CD.