ANIMAL MODEL OF GLOBOID CELL LEUKODYSTROPHY IN RHESUS MONKEYS: LYSOSOMAL STORAGE

恒河猴球状细胞脑白质营养不良的动物模型：溶酶体储存

• 批准号: 6116204
• 负责人: GARY B BASKIN
• 金额: $ 16.14万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6116204
• 关键词: Mammalia; congenital disorders; gene therapy; inborn metabolism disorder; model design /development; nervous system; orphan disease /drug; technology /technique

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. Since then, we 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 by PCR. 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 mutation responsible for GLD in these monkeys consists of the deletion of the AC dinucleotide corresponding to cDNA positions 387 and 388. This results in a frame shift, leading to a premature stop codon. 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. The average activity for 21 normal monkeys was 0.94 nmol/hr/mg of protein, while the average for 20 carriers was 0.53. Psychosine levels in the brains of affected infants were very high. 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. We have performed chorionic villus sampling for 3 birth seasons and have accurately diagnosed the genetic status of the fetuses each year prior to birth. In a related subproject, we have begun preliminary studies to determine the most appropriate way to deliver therapeutic genes to affected monkeys. This nonhuman primate model will be valuable for studies of gene therapy of this and similar disorders. FUNDING Base Grant, Venture Research PUBLICATIONS Luzi P, Rafi MA, Victoria T, Baskin GB, Wenger DA. Characterization of the rhesus monkey galactocerebrosidase (GALC) cDNA and gene, and identification of the mutation causing globoid cell leukodystrophy (Krabbe disease) in this primate. Genomics 42:319-324, 1997; Baskin GB, Ratterree M, Davison BB, Falkenstein KP, Clarke MR, England JD, Vanier MT, Luzi P, Rafi MA, Wenger DA. Genetic Galactocerebrosidase deficiency (globoid cell leukodystrophy, Krabbe disease) in rhesus monkeys. Lab Anim Sci 48:476-

球状细胞脑白质营养不良是一种罕见的常染色体隐性遗传病 B-半乳糖苷酶活性低引起的疾病，B-半乳糖苷酶是一种溶酶体酶 髓磷脂代谢中重要的酶。 一只刚出生的恒河猴 这种疾病于 1989 年在 TRPRC 被诊断出来。从那时起，我们 有意使这组动物近亲繁殖，并于 1996 年观察到两只 其他受影响的婴儿。 这两只纯合受影响的动物 允许我们与 Jefferson Medical 的 David Wenger 博士合作 费城学院，确定致病突变 恒河猴 GALC 基因并明确鉴定 22 种携带者动物 PCR。 恒河猴 GALC cDNA 的序列与 推导的氨基酸序列与人的氨基酸序列有97%的一致性 人类。 这些猴子中导致 GLD 的突变包括 对应于cDNA位置的AC二核苷酸的缺失 387 和 388。这会导致帧移位，导致过早 终止密码子。 在受影响的 2 个纯合子中测量了 GALC 活性， 21只正常猴和20只携带猴。 2 名受影响的婴儿患有 GALC 活动量低于正常值的 2%。 21 正常人的平均活动 猴子的蛋白质含量为 0.94 nmol/hr/mg，而 20 只猴子的平均值为 0.94 nmol/hr/mg。 运营商为0.53。 受影响者大脑中的心理激素水平 婴儿的发病率非常高。 我们建立了成纤维细胞并进行了 EBV 转化 从这些纯合子婴儿中进行淋巴细胞培养，并已开始 含有逆转录病毒载体的体外治疗研究 人类 GALC 基因。 我们已经进行了 3 次绒毛膜绒毛取样 出生季节并准确诊断出出生的遗传状况 每年胎儿出生前。 在一个相关的子项目中，我们有 开始进行初步研究以确定最合适的方法 将治疗基因传递给受影响的猴子。 这种非人类灵长类动物 模型对于研究这种和类似的基因治疗很有价值 失调。 资金基础拨款，风险研究出版物 Luzi P， 拉菲·马、维多利亚·T、巴斯金·GB、温格·DA。 的表征 恒河猴半乳糖脑苷酶（GALC）cDNA和基因，以及 鉴定导致球状细胞脑白质营养不良的突变 （克拉伯病）在这种灵长类动物中。 基因组学 42：319-324，1997；巴斯金 GB、Ratterree M、戴维森 BB、Falkenstein KP、克拉克 MR、英格兰 JD、 Vanier MT、Luzi P、Rafi MA、Wenger DA。 遗传性半乳糖脑苷脂酶 恒河猴缺乏症（球状细胞脑白质营养不良、克拉伯病） 猴子。 实验室动画科学 48:476-