Skin is easily accessible and in principle is an ideal target for gene therapy of inherited skin disorders.However this has not become a reality. This proposed project intends to recapitulate rare inherited skindiseases in organotypic tissue cultures using keratinocytes differentiated from human induced pluripotentstem (iPS) cells into which relevant gene mutations have been introduced. Several labs have recentlyreported the derivation of iPS cells from mouse or human somatic cells. This advance creates a majoropportunity for developing disease models and therapies. Somatic cells are reprogrammed to become iPScells by expressing three chromatin-remodeling transcription factors, Sox2, KLF4, and Oct4 over a period ofabout 3 weeks. Tim Townes' lab has successfully cured mice with human sickle ceil disease using genecorrectediPS cells. Presently, the transgenes are introduced into the somatic cells via retroviruses orlentiviruses. However, mutagenic insertion of these vectors has been a serious concern and it is highlydesirable to develop a non-integrating vector. The short and long term Specific Aims are: (1) To construct anon-integrating plasmid vector to express the transgenes. The replicon is based on the simple replicationrequirements of the human papillomavirus DMA plasmid. The strategy for transgene expression is beingdeveloped by the Townes lab and will be incorporated into our plasmid-based vectors. (2) To transfect vectorDNA into neonatal foreskin fibroblasts and derive iPS cells. (3) To differentiate the iPS cells into thekeratinocyte lineage. The properties of these keratinocytes and their ability to differentiate into squamousepithelium in organotypic cultures will be examined and compared to those of primary neonatal foreskinkeratinocytes. (4) To recapitulate EB-simpfex skin models in vitro. Dominant mutations in keratin 5 or keratin14 genes identified in epidermolysis bullosa simplex patients will be introduced into iPS cells (or humanfibroblasts prior to derivation of iPS cells) by homologous recombination. The iPS cells will be differentiatedinto keratinocytes that will then be used to develop squamous epithelium in organotypic raft cultures andexamined by in situ methods. Success in these experiments would serve as proof-of-principle that iPS cellscan be used widely by researchers to study genetic skin diseases and to test for new therapeuticapproaches.
Showing the most recent 10 out of 228 publications
