Dennis Roop, PhD
Ganna Bilousova
Jiang Chen, MD 
University of Colorado Denver, Aurora, CO

Dennis Roop, PhD	Jiang Chen, MD

Objective:
There is no cure for epidermolytic ichthyosis (EI) (formerly called epidermolytic hyperkeratosis or EHK). Therefore, novel gene therapy approaches become extremely attractive for this inheritable epidermal disease caused by single gene mutations in either keratin 1 or 10. In order to permanently correct epidermal diseases, it is necessary to design a therapeutic approach that is able to correct epidermal stem cells. Preliminary studies with a pre-clinical mouse model for EI indicate the feasibility of ex vivo correction of mouse EI cells followed by reconstitution of the skin in a graft model. Before this approach can be tested in humans, it is desirable to first test this ex vivo gene therapy with human EI cells. For such purposes, large numbers of human EI cells are required.

Relevance to the mission of the Foundation for Ichthyosis & Related Skin Types, Inc.^®
The aim of the proposed projects in this application is to generate EI cell lines so that emerging therapies can be tested on human cells. Any therapy that is proven to be safe and effective using these cells will pave the way for clinical trials for EI patients; thus, meeting the priority of the research program and mission of the Foundation.

UPDATE:   October 2010

figure 1

One of the goals of this project is to generate induced pluripotent stem cells from Epidermolytic ichthyosis (EI) patients. What are induced pluripotent stem (iPS) cells?   iPS cells are reprogrammed adult skin cells that have essentially all of the properties of embryonic stem cells. Embryonic stem cells can give rise to all cell types in the human body, thus they are called pluripotent cells because of this property. The fact that embryonic stem cells are pluripotent has created a lot of excitement for their enorm ous potential to regenerate all types of tissues and revolutionize the treatment of diseases and trauma. The potential use of embryonic stem cells has also been hotly debated over the last decade because of the ethical issues associated with the necessity to destroy early stage embryos to harvest these cells (see fig. 1). The ability to generate iPS cells from a biopsy of adult skin potentially solves many problems associated with the use of embryonic stem (ES) cells. This strategy would not only eliminate the need for generating ES cells from fertilized human embryos, but also avoid the complication of immune rejection, which might occur with the transplant of cells derived from ES cells onto an unrelated individual. Therapies based on the use of iPS cells would truly be “personalized medicine”, since the iPS cells would be generated from the same individual who would ultimately be treated with these cells. The process of reprogramming, or inducing pluripotency, basically involves taking a committed adult skin cell and introducing factors into these cells that are capable of removing all of the cell’s memory and reverting that cell back to an embryonic state, a process analogous to stripping everything stored on the hard drive of a computer.

figure 2

There has been success taking a biopsy from the skin of an EI patient, placing the skin cells in culture, and reprogramming these cells into iPS cells (see fig. 2). Like embryonic stem cells, iPS cells can be grown in culture in the laboratory indefinitely. Now that there is an unlimited supply of iPS cells generated from an EI patient, the goal is to see if the defect can be corrected genetically; in this case a mutation in the keratin 1 gene. There has been recent success differentiating iPS cells into keratinocyte stem cells and showing that these cells can form a normal epidermis when grafted onto mice. Therefore, if the keratin 1 defect in the EI-derived iPS cells can be safely corrected, the ultimate goal would be to differentiate the corrected iPS cells into keratinocyte stem cells and return these cells to the EI patient as a skin graft (see fig. 2).

One of the goals of our FIRST funded project is to generate induced pluripotent stem (iPS) cells from epidermolytic ichthyosis (EI) (formerly called epidermolytic hyperkeratosis) patients. iPS cells are patient-specific reprogrammed adult skin cells that have essentially all of the properties of embryonic stem cells. The process of reprogramming, or inducing pluripotency, basically involves taking a committed adult skin cell and introducing factors into these cells that are capable of removing all of the cell’s memory and reverting that cell back to an embryonic-like state. To obtain the adult skin cells, a biopsy is taken from the skin of an EI patient, and the skin cells are grown in culture. Reprogramming factors are then introduced into the adult skin cells and over time, colonies or clones of iPS cells appear in the culture dish. The advantage of generating patient-specific iPS cells is that these cells can be grown indefinitely in culture and they can be frozen and stored indefinitely for future use.   Having an unlimited supply of patient-specific iPS cells provides us with the opportunity to genetically correct the defect, in the case of EI, mutations in either the keratin 1 (K1) or keratin 10 (K10) gene. We have recently been successful in correcting the defect in the K1 gene in one patient’s iPS cells with a technique that uses zinc finger nucleases. Zinc finger nucleases are like molecular scissors that allow us to open up the K1 gene near the site of the mutation and replace the defective region with a normal region of DNA. We are currently performing tests on the corrected iPS cells to make sure that they are genetically stable. Once this has been confirmed, we will differentiate the corrected iPS cells into keratinocyte stem cells and determine if these cells can form a normal epidermis when grafted onto mice.