Skip to main content

Ex vivo gene therapy for recessive dystrophic epidermlysis bullosa: pre-clinical and clinical studies

Final Report Summary - THERAPEUSKIN (Ex vivo gene therapy for recessive dystrophic epidermlysis bullosa: pre-clinical and clinical studies)

Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe rare inherited skin disorders affecting more than 2 000 patients in Europe. Patients suffer since birth from skin blistering, and from severe local and systemic complications resulting in poor prognosis. Current medical care protocols for RDEB patients are limited to palliative procedures to treat bullous lesions and severe complications of the disease (joint contractures, malnutrition, infections, sepsis and aggressive skin cancer). However, these treatments cannot prevent recurrent blistering arising from defective type VII collagen expression. This creates a tremendous medical, economic and psychological burden for patients through recurrent medical care, hospitalisations and surgical interventions.

The THERAPEUSKIN partners have successfully developed a safe (Self Inactivating, SIN) retroviral vector for genetic transfer of COL7A1 cDNA into keratinocytes and fibroblasts. We have used this vector to genetically correct primary RDEB keratinocytes and fibroblasts in culture, and to generate human skin equivalents suitable for grafting. Using this model, we could demonstrate long-term in vivo expression of recombinant type VII collagen along the dermal epidermal-junction and anchoring fibril formation up to five months after grafting onto nude mice. However, the variability in the quality and yield of the SIN COL7A1 retroviral vector supernatants obtained by tri-transfection, led us to develop a producer clone as a source of viral particles. Such a clone in which COL7A1 cDNA expression was driven by the EF1-a gene promoter was produced in collaboration with the InsertAgene European project. Viral supernatants from this clone showed reproducible high titres and efficient transduction of primary human RDEB keratinocytes and fibroblasts. Transduction of RDEB epidermal stem cells with this COL7A1 vector did not alter their proliferative capacity. Grafting experiments of skin equivalents made of RDEB keratinocytes and fibroblasts genetically corrected with this vector demonstrated long-term functional correction and anchoring fibril formation several months after grafting on nude mice.

However, an important Neomycin contamination in COL7A1 retroviral supernatants due to the weakness of the polyadenylation signal present in the MLV 3' LTR and to the design of the producer clones exchange cassette led us to abandon the COL7A1 producer clone.

The production process of the SIN COL7A1 retroviral vector by tri-transfection was therefore optimised. This resulted in reproducible viral titres with good transduction efficiency, which led us to choose this system for the preparation of the clinical batch by a subcontractor, EUFEST.

To address safety issues relevant to ex vivo gene therapy, we injected subcutaneously genetically corrected RDEB cells into nude mice, and showed no tumourigenic potentiality up to one year after injection. To investigate a possible immune response against type VII collagen, we developed in vitro ELISPOT and ELISA tests, which we validated in patients affected with acquired dystrophic EB resulting from autoantibodies against type VII collagen. We showed that these tests were highly specific and sensitive, which allowed us to use them for RDEB patient selection.

In conclusion, the main objectives of the THERAPEUSKIN project have been achieved:
i. a safe SIN COL7A1 retroviral vector allowing functional and long term expression of type VII collagen in vivo has been designed, produced and tested, demonstrating the feasibility of the approach in a pre-clinical model;
ii. the manufacturing processes for the production of a clinical-grade GMP viral batch have been set up, which will lead to the production of a ready to use, clinical-grade viral batch;
iii. Candidate RDEB patients have been selected and a first phase I/II clinical trial is in preparation.

Important safety aspects have been fulfilled, including prediction of the immune response towards type VII collagen, demonstration of lack of toxicity and tumorigenicity in vivo. An orphan drug designation has been approved by the E.M.E.A. A clinical centre approved by the French regulatory agencies has been identified to perform this clinical trial. The clinical protocol has been validated. The quality of the clinical-grade viral batch will be essential for the feasibility and the safety of this trial. This will allow completion and submission of the mandatory documents to the competent regulatory agency (AFSSAPS) in order to start the clinical trial.

Related documents