The development of human embryonic stem (hES) cell lines that carry a disease causing mutation can provide insight into the mechanisms underlying disease progression as well as into the development of therapies that can ameliorate that pathology. The primary goal of this proposal will be the development of novel hES cell lines from embryos that will manifest a given genetic disease upon further development. This will be achieved following two distinct approaches. The first will be through the identification of embryos that are homozygous for a given mutation and the generation of novel cell lines from these embryos. These embryos will be identified by preimplantation genetic diagnosis (PDG). The afflicted embryos will then be grown according to established protocols known to generate human ES cell lines. The second approach will involve the generation of disease associated homozygote cell lines through a technique that will specifically modify a gene sequence and introduce a disease associated mutation. This technique, small fragment homologous replacement (SFHR), has been shown to be effective at modifying DNA sequences in human cells. SFHR-mediated changes are caused by small DNA fragments (SDFs) that are introduced into the cells. The SDFs are effectively the same as the gene target sequences except for the changes to be introduced. Initial studies will target genes on the X-chromosome of normal male hES cells that carry only one X-chromosome. The genetic diseases that are anticipated to be served by this proposal include, but are not limited to, cystic fibrosis, sickle cell disease, ?- and ?-thalassemia, Duchenne’s and Becker muscular dystrophy, X chromosome-linked severe combined immune deficiency (SCID-X1), spinal muscular atrophy (SMA), Guacher’s disease, Fanconi anemia, and Lesch-Nyhan syndrome.
This proposal will provide benefit to the citizens of California by increase our knowledge of the basis of genetic diseases and by providing a means to develop new and more effective therapies for these diseases. This project is focused on improving the health and well being of the citizens of California and could have far reaching positive implications for health and economic factors influencing the quality of life for the citizens of this state.