No effective treatments are available for epidermolysis bullosa (EB), a group of rare inherited blistering disorders that can be devastating and in some cases lethal. The technological breakthrough that allows adult skin cells to be reprogrammed into induced pluripotent stem cells (iPSCs) now offers the possibility of developing a permanent corrective therapy for EB. The therapeutic approach relies on the generation of patient-specific iPSCs, which then undergo genetic correction and differentiation into skin cells suitable for transplantation. The generation of iPSCs from the same patient in need of treatment would not only potentially avoid the complication of immune rejection, but also provide an unlimited and scalable source of patient- specific cells suitable for transplantation. For the initial studies funded by the parent R01, we focused on developing an iPSC-based therapy for a cohort of patients suffering from the Dowling-Meara subtype of EB simplex (EBS-DM), who share the same R125C ?hot spot mutation? in the keratin 14 (Krt14) protein that is responsible for 70% of the EBS-DM cases. This EBS-DM study has allowed us to address all of the major obstacles and pre-requisites required for the development of a clinically relevant iPSC-based therapy for EBS. Specifically, we have developed a novel, efficient method for the generation of clinically relevant integration- free iPSCs. We have also used non-integrating selection marker-free gene editing technology to successfully correct the genetic defect in iPSCs generated from an EBS patient. In addition, our high-efficiency of reprogramming has recently allowed us to combine gene editing and reprogramming of EBS fibroblasts into a one-step procedure. This revision application is proposing to expand the one-step combined gene editing and reprogramming approach to a more severe form of EB, recessive dystrophic EB (RDEB) and to advance our findings toward a clinical trial. Given that the collaborative efforts among several groups will be required to ensure the successful translation of complex iPSC-based therapies into the clinic, we have recently entered into a consortium with Stanford and Columbia Universities to facilitate the development of an optimal manufacturing protocol for genetically corrected iPSC-derived cell products for RDEB treatment. Therefore, Aim 1 of this proposal is to improve the safety and reduce the manufacturing complexity of an iPSC-based therapy for RDEB by combining reprogramming and gene editing into a one-step procedure;
Aims 2 and 3 are to manufacture cGMP-compliant reagents for reprogramming and gene editing respectively to assist the Consortium in generating Investigational New Drug (IND) - enabling data for the FDA. The EB iPS Cell Consortium?s success in obtaining approval for a clinical trial for RDEB would expand the therapeutic potential of iPSCs for the treatment of a variety of monogenic diseases affecting other internal organs, where the difficulty in monitoring adverse effects of an iPSC-based therapy would make them unlikely first targets.

Public Health Relevance

Epidermolysis bullosa (EB), is a group of rare, incurable, inherited skin blistering diseases that result in severe blistering and scaring, and in some cases, early death. Building on our scientific progress toward the development of a safe, induced pluripotent stem cell (iPSC)-based therapy for EB simplex (EBS), that we achieved during the course of our parent R01, we propose to extend our studies to a more severe type of EB, recessive dystrophic EB (RDEB), and to manufacture clinical grade materials for reprogramming and gene editing for future clinical trials. If the studies outlined in this application result in obtaining approval for a clinical trial for RDEB, these pre-clinical data will pave the way for approval of iPSC-based clinical trials for a variety of other genetic diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
3R01AR059947-05S1
Application #
9516699
Study Section
Special Emphasis Panel (ZHL1)
Program Officer
Cibotti, Ricardo
Project Start
2012-09-17
Project End
2018-08-31
Budget Start
2017-09-21
Budget End
2018-08-31
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Dermatology
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
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Webber, Beau R; O'Connor, Kyle T; McElmurry, Ron T et al. (2017) Rapid generation of Col7a1-/- mouse model of recessive dystrophic epidermolysis bullosa and partial rescue via immunosuppressive dermal mesenchymal stem cells. Lab Invest 97:1218-1224
Boyle, William S; Senger, Kyle; Tolar, Jakub et al. (2017) Heparin Enhances Transfection in Concert with a Trehalose-Based Polycation with Challenging Cell Types. Biomacromolecules 18:56-67
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Agrawal, Pranav; Ingle, Nilesh P; Boyle, William S et al. (2016) Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension. ACS Appl Mater Interfaces 8:8870-4
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Liu, Ying; Snedecor, Elizabeth R; Zhang, Xu et al. (2016) Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75. J Invest Dermatol 136:45-51
Vanden Oever, Michael; Muldoon, Daniel; Mathews, Wendy et al. (2016) miR-29 Regulates Type VII Collagen in Recessive Dystrophic Epidermolysis Bullosa. J Invest Dermatol 136:2013-2021
Tolarova, Marketa; Tolar, Jakub (2015) From Mesoderm to Mesodermatology: Bone Marrow Mesenchymal Cells Heal Skin Wounds. Mol Ther 23:1283-1284
Nevala-Plagemann, Christopher; Lee, Catherine; Tolar, Jakub (2015) Placenta-based therapies for the treatment of epidermolysis bullosa. Cytotherapy 17:786-795

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