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.

球状细胞白质营养不良是一种罕见的常染色体隐性遗传 由溶酶体B-半乳糖苷酶活性低水平引起的疾病 髓鞘代谢中重要的酶。一只幼年恒河猴 这种疾病是1989年在TRPRC被诊断出来的。在这中间的七个人中 多年来，我们故意对这群动物进行近亲繁殖，并在1996年 观察了另外两名受影响的婴儿。这两个纯合子 受感染的动物使我们在大卫·温格博士的合作下， 费城杰斐逊医学院，以确定 恒河猴GALC基因的致病突变和明确的 找出22种携带病毒的动物。恒河猴GALC基因的序列为 与人类的同源性为98%，推导的氨基酸序列为97%。 与人类一模一样。人类和恒河猴的组织 GALC基因也很相似。GLD的致病基因突变 这些猴子被发现由AC的缺失组成 对应于cDNA387和388位的二核苷酸。这 突变会导致平移过程中的帧移动，从而导致 删除后终止密码子46个碱基对。使用基于聚合酶链式反应的技术，我们 能够识别出22个携带者和21个非携带者 一群繁殖的猴子。携带者由四名成年男性组成， 八只成年雌性和十只幼崽。GALC活性是在 纯合子感染2只，正常21只，携带者20只。这两个 受影响的婴儿GALC活性不到正常的2% 白细胞和培养的皮肤成纤维细胞。21年的平均活动量 正常恒河猴的蛋白质含量为0.94nmol/小时/毫克，而 20家航空公司的平均水平为0.53。我们建立了成纤维细胞和 这些纯合子婴儿的EBV转化的淋巴细胞培养，以及 已经开始了逆转录病毒载体的体外治疗研究 含有人类GALC基因。尽管这个动物模型将会是 对有关生物化学和发病机制的研究是有用的 GLD，非人类灵长类动物模型将是最有价值的 这种疾病和类似疾病的基因治疗。几家航空公司正在 目前正在怀孕，并在宫内诊断和治疗这些 婴儿期正在进行中。