Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system

Background Canavan disease (CD) is an autosomal recessive leukodystrophy ch aracterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retarda tion and early death. Gene therapy has been attempted in a number of childr en with CD. The lack of an animal model has been a limiting factor in devel oping vectors for the treatment of CD. This paper reports the successful cr eation of a knock-out mouse for Canavan disease that can be used for gene t ransfer. Methods Genomic library lambda knock-out shuttle (lambda KOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid wi th 10 base pair (bp) deletion of exon four of the murine aspa. Following li nearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and co nfirmed by Southern analysis. Chimeras were generated by injection of ES ce lls to blastocysts. Germ line transmission was achieved by the birth of het erozygous mice as confirmed by Southern analysis. Results Heterozygous mice born following these experiments have no overt ph enotype. 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 ne rvous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data g enerated from this animal model will be useful for developing strategies fo r gene therapy in other neurodegenerative diseases. Copyright (C) 2000 John Wiley & Sons, Ltd.