Abstract
Dystrophic epidermolysis bullosa (EB) is a serious genetic skin blistering condition. There are several manifestations of this disorder, each corresponding to a difference in severity of the same symptoms. The least prevalent form of epidermolysis bullosa is termed autosomal recessive dystrophic epidermolysis bullosa, but is symptomatically the most severe. The condition is the result of a mutant collagen gene, COL7A1, which renders a person incapable of producing proteins that collectively form type VII collagen. Collagen is produced and secreted from keratinocytes in the skin and plays a quintessential role in anchoring the basal lamina to the dermis and epidermis. Without proper expression of the COL7A1 gene, the skin is incapable of anchoring to the underlying tissue resulting in particularly fragile skin that can be easily damaged by minor friction or even routine tasks such as eating. Currently, there is no cure for autosomal recessive dystrophic epidermolysis bullosa, however, advancements in induced pluripotent stem cell (iPSC) technology offers hope for future treatments. There are many advantages in using iPSCs in EB patient treatment, two of the advantages being the evasion of graft rejection as well as avoiding ethical issues surrounding the use of human embryonic stem cells. By applying patient iPSCs, a patient’s own tissue can be engineered, and human embryos to generate pluripotent stem cells are not required. The process of generating iPSCs begins with the reprogramming of mature skin cells by the application of an integrating, but also excisable lentiviral vector. The vector delivers four genes, Oct4, Sox2, c-Myc, and Klf4 into the skin cell’s nucleus. The addition of these four genes causes the adult cell to express genes normally only expressed during embryonic cell development. At this stage, the reprogrammed cell has properties similar to that of an embryonic stem cell and has the potential to differentiate into any tissue type. The integrated reprogramming vector, however must be removed in order to produce a clinical grade product. To accomplish this, a LoxP / Cre recombinase approach is used. Cre recombinase is added to established iPSC colonies to excise the vector. Following vector excision a DNA plasmid targeting vector with the fully functional COL7A1 gene is introduced into the iPSCs. As the cells divide, the chance arises for homologous recombination to occur, the functional gene then replaces the mutant gene in the target locus. While the efficiency of homologous recombination is only between 2-10% we can select for successfully corrected iPSCs and expand these cells to provide a relevant number of gene corrected pluripotent cells for clinical use. The corrected cells are then differentiated into keratinocytes and finally manufactured into dermal grafts which can be used for transplantation onto the patient. While many of the complicated steps involved in the generation of a clinical prod are demonstrated in this project, the primary objective revolves around the differentiation assay. This is one of the final steps in the entire process in the generation of clinical grade keratinocytes, and currently the least developed process. Our partners at Stanford University have routinely generated EB fibroblast derived iPSCs, excised the lentiviral vector, and corrected the cells via homologous recombination; however, they have not performed the differentiation and purification process required for the final keratinocyte population starting with iPSCs. A theoretical protocol has been generated by the Stanford Oro group for the derivation of keratinocytes from H9 hESCs. The task for this research project has been primarily to elucidate the efficacy of translating the Oro protocol from H9 hESC differentiation to iPSC differentiation. Furthermore, this project has been challenged with the task of adapting the original protocol in any way necessary resulting in a functional protocol for the reproducible differentiation of iPSCs into functional keratinocytes. This project took place during the second year of four years of total CIRM funding for the EB project. During this time, remarkable progress has been made and the vast majority of the objectives have been met. At the time of completion of this work, the entire EB project is nearing the investigational new drug (IND) application phase which will represent an important milestone for the cause of novel EB therapy research as well as the field of regenerative medicine.