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Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system
R Matalon1, P L Rady, K A Platt
1Department of Pediatrics, Children's Hospital, UTMB Galveston, TX 77555-0359, USA. rmatalon@utmb.edu
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 lambda knock-out shuttle (lambdaKOS) 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.
Insights
Researchers created a new mouse model for Canavan disease (CD), a genetic disorder affecting brain white matter. This ASPA-deficient mouse exhibits key CD symptoms and will aid in developing gene therapies for this and other neurodegenerative diseases.
Area of Science:
- Genetics
- Neuroscience
- Animal Models
Background:
- Canavan disease (CD) is a fatal autosomal recessive leukodystrophy.
- It is caused by aspartoacylase (ASPA) deficiency, leading to elevated N-acetylaspartic acid (NAA) levels.
- A lack of suitable animal models has hindered the development of gene therapies for CD.
Purpose of the Study:
- To create a genetically engineered mouse model for Canavan disease.
- To establish a platform for testing gene transfer vectors and understanding CD pathophysiology.
- To facilitate the development of novel therapeutic strategies for neurodegenerative diseases.
Main Methods:
- A knock-out strategy was employed using a lambda knock-out shuttle (lambdaKOS) system.
- A plasmid with a 10 bp deletion in exon four of the murine aspa gene was created.
- ES cells were targeted, and chimeras were generated for germline transmission, resulting in heterozygous and homozygous mice.
Main Results:
- Homozygous mice exhibit neurological impairment, macrocephaly, and white matter disease.
- ASPA activity is deficient, and NAA levels are elevated in the urine of homozygous mice.
- MRI and MRS confirm white matter changes and elevated NAA levels in the brains of affected mice.
Conclusions:
- The developed ASPA-deficient mouse serves as a crucial animal model for Canavan disease.
- This model is instrumental for advancing gene transfer vector development for CD treatment.
- It offers a valuable tool for studying CNS-specific gene therapy and neurodegenerative disease mechanisms.