Matalon R, Rady P L, Platt K A, Skinner H B, Quast M J, Campbell G A, Matalon K, Ceci J D, Tyring S K, Nehls M, Surendran S, Wei J, Ezell E L, Szucs S
Department of Pediatrics, Children's Hospital, UTMB Galveston, TX 77555-0359, USA.
J Gene Med. 2000 May-Jun;2(3):165-75. doi: 10.1002/(SICI)1521-2254(200005/06)2:3<165::AID-JGM107>3.0.CO;2-R.
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.
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.
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.
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.
卡纳万病（CD）是一种常染色体隐性遗传性脑白质营养不良症，其特征为天冬氨酸酰基转移酶（ASPA）缺乏，且脑和体液中N-乙酰天门冬氨酸（NAA）水平升高，伴有严重智力发育迟缓并早亡。已尝试对一些患有卡纳万病的儿童进行基因治疗。缺乏动物模型一直是开发用于治疗卡纳万病的载体的一个限制因素。本文报道成功创建了一种可用于基因转移的卡纳万病基因敲除小鼠。
筛选基因组文库λ基因敲除穿梭载体（lambdaKOS），分离出特定的pKOS/Aspa克隆，并用于构建一个质粒，该质粒使小鼠aspa基因第四外显子缺失10个碱基对（bp）。线性化后，将该质粒电穿孔导入胚胎干细胞（ES细胞）。经正负筛选鉴定出正确靶向的ES细胞克隆，并通过Southern分析进行确认。通过将ES细胞注射到囊胚中产生嵌合体。经Southern分析确认杂合子小鼠的出生实现了种系传递。
这些实验后出生的杂合子小鼠没有明显的表型。纯合子小鼠表现出神经功能障碍、巨头症、全身性白质疾病、ASPA活性缺乏以及尿液中NAA水平升高。纯合子小鼠脑部的磁共振成像（MRI）和波谱分析（MRS）显示出卡纳万病特有的白质变化以及NAA水平升高。
新创建的ASPA缺陷小鼠建立了一种重要的卡纳万病动物模型。该模型对于开发用于治疗卡纳万病的基因转移载体应是有用的。用于中枢神经系统（CNS）以及调节脑中NAA水平 的载体应能进一步增进对卡纳万病病理生理学的理解。从该动物模型产生的数据将有助于开发针对其他神经退行性疾病的基因治疗策略。
