Revertant Cell Therapy

Antoni Gostynski, Groningen

Revertant mosaicism

Revertant cell therapy is a novel therapeutic possibility in patients with epidermolysis bullosa (EB) and revertant mosaicism (also called "natural gene therapy") - a phenomenon of a second genetic event, i.e. a back mutation/reversion, which restores function of the disease-causing gene. Revertant mosaicism has been described for a number of diseases of blood, muscle, liver and skin.  In the skin it was first demonstrated for the junctional type of EB. In a patient with mutations in the type XVII collagen (COL17A1) gene – healthy skin patches were present next to affected skin. It was also described later for other types of EB.

Concept of revertant cell therapy

Presence of revertant (=healthy) skin patches has a great value for EB patients. These are however limitations in area and control over the placement of the patches on one's body. Because of it, the idea of revertant cell therapy was born: to expand revertant patches by transplantation of the patient's own skin cells (=keratinocytes) in a form of engineered skin grafts.

An elegant approach would be to process a small skin biopsy (about 1cm2), isolate skin cells, and culture them in a laboratory setting. Stem cells of the skin, present in such a biopsy, would proliferate and expand, and skin grafts of an area much greater than the original biopsy (theoretically up to few m2) could be produced. Those revertant skin grafts could then be transplanted back to the patient and provide long-term improvement.

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Clinical experience with revertant cell therapy

First experiments with revertant keratinocytes showed a possible problem with mutant (=unhealthy) cells overgrowing healthy cells during the graft production. The produced skin graft consisted of only 3% revertant cells; the other 97% being patient's mutant cells. Such a low percentage of revertant cells is not sufficient to maintain a healthy skin patch.

To investigate this problem we have used an animal model and analysed every step of revertant cell therapy: cell proliferation, graft production, transplantation and long-term survival. Unfortunately, we have observed a gradual disappearance of type COL17A1 revertant cells during the whole process (from 40 to 3 %). The reason for this disadvantage of revertant cells during the whole process is yet to be established. Currently we are assessing possibilities of revertant cell therapy for revertant patients with dystrophic EB. In the preliminary studies we see a higher rate of revertant cell survival than in previous experiments.

Future and chances for successful therapy

There are a few possible explanations why the revertant cell therapy has not yet been successful: the small number of revertant stem cells, growth disadvantage outside the patient's body or unwanted influence of culture techniques on revertant cells. We are currently looking into possible cell isolation and selection methods to enrich our grafts with revertant cells.

In the near future the need for enrichment might be replaced by revertant cell therapy based on induced pluripotent cells (iPS). Those cells, induced from patient's revertant cells, could be differentiated back into keratinocytes and used to produce multiple skin grafts, or into bone marrow cells for systemic therapy. With these new possibilities we are eager to continue our experiments.

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