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We continue our efforts to understand how bone marrow transplant (BMT) for recessive dystrophic epidermolysis bullosa works and how to make it safer as a systemic therapy. In parallel, we are investigating gene therapy strategies and gathering the safety and efficacy data needed to submit a proposal to the FDA. We are working with different transplant strategies, using haploidentical (e.g., parent or sibling) grafts, serial infusions of donated mesenchymal stromal cells, and several methods of skin grafting using skin cells from the healthy BMT donor, creating skin from gene-corrected or mosaic (naturally healthy) patient cells, and 3D printing of patient-specific skin.
Other questions we are researching include:
· Which cells migrate to the skin?
· What drives the relative contribution of recipient and donor C7?
· What effect does pre-transplant hospital care have?
· What are the side effects of pre-transplant conditioning?
· What factors predict how effective the response is to BMT?
“Our goal is to create a toolbox of treatments that could be individualized for a patient based on factors like their age, overall health, and genetic mutation. These treatments would be able to address the internal, as well as the external, damage done by EB and return the patient to health.”
This year we have continued to make progress on the steps needed to make several cell therapies available in the clinic for RDEB patients. This year we were able to:
In the year to come, we will continue to evaluate and incorporate advances throughout the field of biomedicine as they are relevant to RDEB.
One example is that we have been able to adapt a new type of gene-editing technology (CRISPR/Cas9) for use with type VII collagen mutations. This advance may give us more ways to use gene editing and may even decrease the costs involved. On the other hand, we are not yet certain about the safety of this technology, so we and others will be working to define the best balance between the treatment benefit and risk.
We also plan to isolate self-corrected skin cells from patches of individuals with RDEB who have areas of unaffected, unblistered skin, and work to expand them to clinically meaningful numbers.
We continue to work on ways to improve expression of the type VII collagen gene in individuals who produce low levels of mutant collagen VII, which could perhaps be stimulated to produce enough collagen to maintain skin integrity.
Perhaps most critically, we will be evolving experimental processes for gene editing and cell culture into robust, simple, reliable techniques (SOPs) that can be easily reproduced in a good manufacturing practices setting, which is a critical step in making these cellular materials available for clinical use.
RESEARCH SUMMARY: (2012/2013)
Jakub Tolar, M.D., Ph.D., part of the trailblazing University of Minnesota team offering experimental but risky blood and marrow transplants aimed at curing Epidermolysis Bullosa (EB), is pursuing innovative research focused on finding a safer treatment alternative for children afflicted with the disease.
One “natural gene therapy” approach involves taking advantage of healthy patches of skin that are present on some children with EB and removing healthy skin cells, turning them into powerful stem cells called induced pluripotent stem cells (or iPS cells, which can be coaxed into becoming any type of cell in the body), expanding them in the lab, and giving them back to the patient where they would grow more healthy skin.
We have identified and created cells that can be corrected. We needed cells, like embryonic stem cells, that had the ability to differentiate into many different cell types. Induced pluripotent stem cells (iPS cells) are adult stem cells that have been reprogrammed to resemble embryonic stem cells in this way. However, the cells that work well to create iPS cells are rare in EB children, and it was not known if iPS cells could be developed from these patients. We were the first to take skin cells (fibroblasts and keratinocytes) from children with recessive dystrophic EB (RDEB) and junctional EB (JEB) and successfully create iPS cells. These personalized iPS cells are capable of making skin and blood cells.
Another approach, “seamless gene therapy,” involves the removal of a patient’s skin cells, “cutting” the DNA at the mutation to correct the gene in the lab, “growing” those corrected iPS cells into blood-forming or skin cells, and reintroducing them into the child’s body.
We are exploring ways to correct these patient-derived iPS cells and have them produce either type VII collagen (for RDEB) or laminin 332 (for JEB). Of the two experimental strategies we are using, the more promising uses enzymes to perform surgery on the genome itself, chemically cutting the DNA in the exact spot of the error, removing the section containing the error, and replacing it with a corrected DNA sequence. Once this is consistently operational in human iPS cells, we will take the next step, which is to test these cells in mice to prove that the repaired cells are both effective and safe. If that is proven in the mouse models, the plan is to correct and restore cells in patients with EB, which promises to offer a lasting treatment and cure for the this disease
We are also pursuing stem cell treatments that could improve the lives of EB patients in the meantime. We are working to identify which cells know to go to the site of an injury and what triggers the repair process. When identified, these cells can be given to patients in greater concentrations, thus supporting the processes that heal the skin.
Over the last three years the faculty of the University of Minnesota Division of Pediatric Blood and Marrow Transplantation have made substantive progress in the use of stem cells in the treatment of individuals with recessive dystrophic epidermolysis bullosa (RDEB).
Research began in 2007. using a mouse model of RDEB. Many different types of cells were tested and their impact on EB examined. The unexpected result of this research was that the mice could be partially treated with a rare subpopulation of bone marrow stem cells rather than skin specific or multipotent stem cells. The principal findings in the mouse model were that donor cells migrate to the skin lesions observed in RDEB, collagen type VII increases rapidly with subsequent development of anchoring fibrils, skin integrity is improved with relative resistance to blister formation in response to trauma, and survival is extended. These discoveries were reported in the journal Blood in 2008.
As a result of this finding in the animal model, a ‘first-in-human’ clinical trial was initiated using unfiltered marrow stem cells in the treatment of children with RDEB. The primary goals were to determine the safety of the treatment and demonstrate enhanced skin and mucosal integrity and resistance to blister formation over time. Between 2007 and 2010, 7 patients with RDEB were enrolled and 6 received a stem cell infusion. The results of this first study in RDEB mimicked the results in the animal model in that donor cells migrated to the skin and mucosa, collagen type VII increased and skin integrity improved in varying degrees over time with increasing resistance to blister formation in response to negative pressure in those that have been studied. Because of the novelty of the study and current outcomes, the results of the clinical trial will be published in the New England Journal of Medicine on August 12, 2010. To the best of our knowledge, this is the first treatment strategy with the potential to reach all injured sites in the skin and mucosa of children with RDEB.
Already the investigators at the University of Minnesota have moved to a second trial, evaluating the potential added benefit of co-infusing mesenchymal stromal cells along with the marrow stem cells. The first patient was enrolled in February 2010. To date, 5 patients (4 with RDEB and 1 with JEB) have been treated with a sixth (RDEB) and seventh (JEB) in the queue. This is the first US trial with an active FDA IND file.
Tolar and Wagner are now attempting to identify the exact stem cell population that can home to and repair the damaged skin in RDEB. Once identified, strategies to isolate and expand these stem cells will be developed with the idea that such a strategy would be used in the future for treating patients. This work is funded by the Epidermolysis Bullosa Medical Research Foundation (EBMRF). EBMRF is proud to sponsor this work that may one day improve the safety and effectiveness of stem cells in the treatment of the more severe forms of EB.