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Analysis of immune responses to ex vivo therapy of EB

Project lead Assoc. Prof Iris Gratz
Organisation EB House Austria, Salzburg, AUSTRIA
Project budget EUR 260,000.00
Start date / Duration 01. Jan 2014 / 24 months
Funder(s) / Co-Funder(s) DEBRA Austria
Research area Molecular therapy, Cellular therapy, Immunology

Project details

Short lay summary

Ex vivo gene therapy has emerged as a potential treatment for EB. However, the neo-antigen introduced by this therapeutic option could induce adverse immune reactivity, especially in protein-deficient patients. To date several reports on the clinical outcome of gene therapy have been published, however, detailed immunological studies are lacking.

Here, we analysed blood and skin samples of a patient suffering from junctional epidermolysis bullosa (JEB) that underwent ex vivo therapy and studied the patient’s immune response.

We hypothesized that JEB patients who show residual full-length protein prior to gene therapy would display immune tolerance towards Lam-332. Additionally, we sought to elucidate whether immune surveillance in the transplanted skin would be restored upon placement of a skin graft that is initially devoid of any immune cells.

Scientific summary

We aimed to analyse the patient’s humoral and cellular response against Lam-332 as well as immune cells infiltrating the skin graft throughout the one-year follow-up after ex vivo gene therapy (i.e. retroviral transduction of epidermal stem cells containing corrected LAMB3 gene followed by grafting of corrected epidermal sheets). We found that the patient’s peripheral blood mononuclear cells (PBMC) did not proliferate ex vivo in response to Lam-332-protein before and after grafting. In addition, T cells did not produce the pro-inflammatory cytokines (e.g. IL-17A, IFN-g and IL-4 in response to Lam-332) and the patient did not form Lam-332-specific antibodies.

In addition to these studies of the specific immune response, we wished to immunologically characterize the engineered skin graft. Healthy human skin is an immunologically rich tissue that is populated by a plethora of immune cells. In contrast, the gene-therapeutically engineered skin tissue grafted in our study initially consisted of a thin layer of gene-therapeutically corrected keratinocytes. We found that We detected functional T cell populations CD4+ and CD8+ T cells that produced IFN-g and IL-17 in the graft within one month after grafting, consistent with an inflammatory response. However, these T cell populations decreased over time and we are currently studying the specific requirements for T cell maintenance and the maintenance of immune surveillance in human skin.

Strategic relevance

  • DEBRA strategic goal: molecular and cellular therapies for EB have the inherent risk of eliciting detrimental immune reactions. Our work contributes to the safety of these therapies.
  • Project goal: to investigate the immune reactions of EB patients in response to EB therapy.
  • Preceding/ follow-on projects, and related projects: We are currently preparing a grant application to further elucidate the role of specific skin resident immune cells in the maintenance of skin T cells as well as skin immune surveillance.

What did this project achieve?

We confirmed our ingoing hypothesis that a patient with a residual protein would not mount a specific response against the gene-therapeutically added protein because it is not truly a neo-antigen (i.e. not immunologically “new”). These results suggest that ex vivo JEB gene therapy is a safe therapeutic approach to restore wild-type protein levels in patients that lack pre-existing immune reactivity and express residual full-length protein.

However, we have obtained data that indicate that the long-term maintenance of specific immune cell populations in the graft might be compromised. We and others have found that adaptive immune cells, such as T cells, and skin resident antigen-presenting cells localize around hair follicles. We hypothesize that molecules provided by adnexal structures such as hair follicles are supporting the long-term maintenance of tissue-resident memory T cells and we will further elucidate this using a novel skin-humanized mouse model.

During this project we have also developed the immunological methods to study the immune response of RDEB patients undergoing ex vivo gene therapy to restore the expression of type VII collagen. We are currently doing this in a phase I/II clinical study performed by the EB House Austria in full collaboration with Prof. Michele De Luca (time line 2017 – 2020).


T cells
EB Immunology
Skin immune Surveillance
EB House
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