ANIMAL MODEL OF GLOBOID CELL LEUKODYSTROPHY IN RHESUS MONKEYS

恒河猴球状细胞脑白质营养不良动物模型

• 批准号: 6247288
• 负责人: GARY B BASKIN
• 金额: $ 6.02万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-09 至 1998-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6247288
• 关键词: Mammalia; Primates; congenital disorders; gene therapy; genetics; model design /development; nervous system; orphan disease /drug; technology /technique development

Globoid cell leukodystrophy is a rare autosomal recessive genetic disease caused by low levels of B-galactosidase activity, a lysosomal enzyme important in myelin metabolism. An infant rhesus monkey with this disease was diagnosed at TRPRC in 1989. In the intervening seven years, we have intentionally inbred this group of animals and in 1996 observed two additional affected infants. These two homozygous affected animals allowed us, in collaboration with Dr. David Wenger, Jefferson Medical College, Philadelphia, to identify the disease-causing mutation in the rhesus GALC gene and to unequivocally identify 22 carrier animals. The sequence of the rhesus GALC cDNA is 98% identical to the human, and the deduced amino acid sequence is 97% identical to that of humans. The organization of the human and rhesus GALC genes are also similar. The mutation responsible for GLD in these monkeys was found to consist of the deletion of the AC dinucleotide corresponding to cDNA positions 387 and 388. This mutation results in a frame shift during translation, leading to a stop codon 46 bp after the deletion. Using a PCR-based technique, we were able to identify 22 carriers and 21 noncarriers amongst the breeding group of monkeys. The carriers consist of four adult males, eight adult females, and ten juveniles. GALC activity was measured in the 2 homozygous affected, 21 normal and 20 carrier monkeys. The 2 affected infants had a GALC activity less than 2% of normal in leukocytes and cultured skin fibroblasts. The average activity for 21 normal rhesus monkeys was 0.94 nmol/hr/mg of protein, while the average for 20 carriers was 0.53. We established fibroblast and EBV-transformed lymphocyte cultures from these homozygous infants, and have initiated in vitro therapeutic studies with retroviral vectors containing the human GALC gene. Although this animal model will be useful for studies concerning the biochemistry and pathogenesis of GLD, the nonhuman primate model will be most valuable for studies of gene therapy of this and similar disorders. Several carriers are currently pregnant, and in utero diagnosis and therapy of these infants is underway.

球形细胞白细胞营养不良是一种罕见的常染色体隐性遗传 由低水平的B-半乳糖苷酶活性（溶酶体）引起的疾病 酶在髓磷脂代谢中很重要。 一个婴儿的恒河猴与 这种疾病于1989年在TRPRC诊断出来。 几年，我们故意掩盖了这群动物，并在1996年 观察到另外两个受影响的婴儿。 这两个纯合子 受影响的动物允许我们与David Wenger博士合作 费城杰斐逊医学院，以确定 恒河galc基因中引起疾病​​的突变并明确地进行 识别22种载体动物。 恒河galc cDNA的序列是 98％与人相同，推导的氨基酸序列为97％ 与人类相同。 人类和恒河组织的组织 galc基因也相似。 负责GLD的突变 发现这些猴子由AC的缺失组成 对应于cDNA位置387和388的二核苷酸。这 突变导致翻译过程中的框架移动，导致 删除后停止密码子46 bp。 使用基于PCR的技术，我们 能够在 猴子的育种群体。 载体由四名成年男性组成， 八名成年女性和十名少年。 GALC活性在 这2个纯合子影响了21个正常和20个载体猴子。 2 受影响的婴儿的GALC活性小于正常的2％ 白细胞和培养的皮肤成纤维细胞。 21的平均活动 正常的恒河猴为0.94 nmol/hr/mg的蛋白质，而 20个载体的平均值为0.53。 我们建立了成纤维细胞和 来自这些纯合婴儿的EBV转化的淋巴细胞培养物， 已经通过逆转录病毒载体开始了体外治疗研究 包含人类galc基因。 虽然这种动物模型将是 对于有关生物化学和发病机理的研究很有用 GLD，非人类灵长类动物模型将对研究最有价值 这种和类似疾病的基因治疗。 几个载体是 目前怀孕，在子宫诊断和治疗中 婴儿正在进行中。