DEVELOPMENT OF A MODEL FOR TAY-SACHS DISEASE

泰-萨克斯病，一种溶酶体蓄积性疾病，主要影响 神经系统，已经在分子水平上进行了表征并发现 与酶的阿尔法链缺陷或异常有关 己糖氨酸酶(己糖氨酸酶A)。编码基因的突变 己糖苷酶A(HEXA)已被发现，在人类中，导致 婴儿期、青春期或成人期起病 泰-萨克斯病。 新的治疗干预方法的发展 这种毁灭性的疾病由于缺乏足够的动物而受到阻碍。 评估新治疗方法疗效的模型， 包括基因治疗。修饰小鼠特定基因的能力 胚胎干细胞利用同源重组、组合 利用修饰的ES细胞促进形成的能力 胚胎-囊胚嵌合体中的生殖细胞的研究，开辟了一个新的场所 用它来建立人类疾病的动物模型。因此， 目前的应用建议开发一种婴儿的小鼠模型， 由一种新的同源基因引起的青少年和成人起病的泰-萨克斯病 重组程序。 本应用程序的总体目标包括：1)开发 HEXA失活建立婴幼儿泰-萨克斯病小鼠模型 胚胎干细胞中的基因。基因的失活将通过以下方式完成 导致NEO插入的同源重组事件和 HSV-tk基因外显子6；2)新同源基因的开发 在特定区域引入细微突变的重组程序 HEXA基因。这一名为“点击并切换”的新程序将被 用于将甘氨酸引入269位的Ser氨基酸取代 在HEXA基因的外显子7。修改后，ES细胞将用于 建立成年型Tay-Sachs小鼠模型；3)利用 “点击并切换”同源重组程序引入甘氨酸 外显子7第250位天冬氨酸氨基酸替换 外显子7的纯合子修饰将作为青少年模型 Tay-Sachs；4)用光和光描述所产生的动物模型 电子显微镜检查的类型、严重程度和发病时间 神经细胞异常的出现。此外， 基因修饰对己糖氨酸酶A活性的影响及其积累 神经系统中单个神经节苷脂的含量将被确定。

Tay-Sachs disease, a lysosomal storage disease affecting primarily the nervous system, has been characterized at the molecular levels and found to be associated with a deficient or abnormal alpha chain of the enzyme hexosaminidase (hexosaminidase A). Mutations in the gene encoding hexosaminidase A (HEXA) have been identified that, in humans, result in the development of the infantile, juvenile, or the adult onset form of Tay-Sachs disease. The development of new methods of therapeutic intervention to alleviate this devastating disease has been hampered by the lack of adequate animal models on which to evaluate the efficacy of new treatment methods, including gene therapy. The ability to modify specific genes in mouse embryonic stem cells by the use of homologous recombination, combined with the ability of the modified ES cells to contribute to the formation of the germ cells in ES-blastocyst chimeras, has opened up a new venue with which to generate animal models of human diseases. Accordingly, the current application proposes to develop a mouse model of infantile, juvenile, and adult-onset Tay-Sachs disease by a novel homologous recombination procedure. The overall objectives of this application include: 1) development of a mouse model of infantile Tay-Sachs disease by inactivation of the HEXA gene in ES cells. The inactivation of the gene will be accomplished by a homologous recombination event resulting in insertion of the neo and hsv-tk genes in exon 6; 2) development of a novel homologous recombination procedure to introduce subtle mutations in specific region of the HEXA gene. This novel procedure, named "hit and switch", will be used to introduce a Gly to Ser amino acid substitution at position 269 in exon 7 of the HEXA gene. Once modified, ES cells will be used to generate a mouse model of adult-onset Tay-Sachs; 3) utilization of the "hit and switch" homologous recombination procedure to introduce an Gly to Asp amino acid substitution at position 250 in exon 7. Animals homozygous for the exon 7 modification will serve as models of juvenile Tay-Sachs; 4) characterization of generated animal models by light and electron microscopy in terms of type, degree of severity, and the timing of appearance of neuronal abnormalities. Additionally, the effects of gene modification on hexosaminidase A activity, and on the accumulation of individual gangliosides in the nervous system will be determined.