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.
