Gerhard Bauer Laboratory, University of California, DavisEpidermolysis bullosa (EB) is a genetic disease in which the skin is not properly anchored to the underlying layers resulting in blister formation due to minor mechanical friction. There are many different manifestation of EB, ranging from mild to lethal. Recessive dystrophic EB, one of the most severe manifestations of this disease is a result of a mutation in the COL7A1 gene which prevents the production of functional collagen 7, a crucial protein in anchoring the basal lamina of the skin to the dermis and epidermis. Since this condition is caused by a known gene mutation, it may be amenable by gene therapy, and has therefore been the subject of many gene therapy approaches, even resulting in clinical gene therapy trials trying to correct somatic cells by the use of retroviral vectors transferring the COL7A1 gene. This is a very difficult undertaking since the correction of all or the vast majority of keratinocytes within a patient’s skin is required to provide reliable anchoring. To address the issue of providing gene correction of the vast majority of a patient’s keratinocytes, our approach is not focused on the correction of somatic cells, but on the correction of induced pluripotent stem cells (iPSCs) generated from a patient’s fibroblasts. Theoretically, an iPSC can be gene corrected and expanded vastly, with subsequent differentiation into gene corrected keratinocytes for grafting onto the patient. For our approach, fibroblasts from and EB patient were exposed to a lentiviral vector containing four known reprogramming factors to result in the generation of iPSCs to serve as a cell source. Clinical grade iPSCs, however, can only be generated if the lentiviral vector, which is integrated into the genome of the target cell, can be removed. Therefore, the vector also transfers lox-p sites, which can be recognized by cre-recombinase and in turn excises the integrated vector DNA. During the culturing of these iPSCs, a DNA targeting vector containing the correct COL7A1 is then added to the cells and through homologous recombination during cell division, the disease causing gene can be removed, and exchanged for the correct COL7A1 gene. These corrected iPSCs can then be directed towards karatinocyte differentiation which may be further manufactured into collagen 7 producing dermal graphs for transplantation onto the patient. We have successfully generated GMP grade iPSCs from patients with EB, have scaled these iPSCs to a clinically useful level and have differentiated them successfully into keratinocytes so a clinical grade keratinocyte graft can be manufactured. We are currently working on the improving the homologous recombination technique required to generate COL7A1 corrected iPSCs at large scale, along with optimizing the directed differentiation towards keratinocytes.