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ANR funded project

Programme de Recherche Translationnelle en Santé (PRTS)
Edition 2013


Validation of PRIMA-1MET and TALEN as a therapeutic tools for visual deficient EEC syndrome

Visual treatment of patients affected by p63 ectodermal dysplasia
We aim at evaluating in vitro (3D culture of patient cells) and in vivo (animal model of EEC) the ability of the drug PRIMA-1 to restore visual deficiency of patients affected by EEC ectodermal dysplasia syndrome.

Validation of the first treatment for this rare disease
Patients affected by ectodermal dysplasia syndrome linked to p63 (EEC) suffer from limb and skin defects, associated with progressive opacification of the cornea that leads eventually to loss of vision.There is no available treatment today. Our network has demonstrated that patient fibroblasts, reprogrammed to iPSC and differentiated into corneal epithelial cells recapitulate the pathology. This unique model allowed us to identify PRIMA-1 as small compound able to correct in vitro the most frequent mutation (R304W). Our objective is to validate PRIMA as theraprutic tool for EEC. For that purpose, PRIMA will be challenged in both 3D organotypic reconstitution assay and in vivo on EEC animal model produced by grafting EEC differentiated cells. Occular toxicity of PRIMA-containing eye drops will be tested in vitro and in vivo as well.

Organotypic reconstruction and animal model to validate the potential of PRIMA-1
EEC-derived reprogrammed induced pluripotent stem cells (EEC-iPSC) and controls will be induced to differentiate into corneal epithelium (+/- PRIMA), grow an air-liquid interface for 3D cornea production in vitro. The formation of a pluristratified epithelium by EEC cells treated with PRIMA will be evalutated by histology and immunostaining. In parallel, an EEC animal model will be created by grating committed EEC-iPSC onto wounded mouse limbus and PRIMA-1MET will be dropped onto defective eyes. These methodologies are already used by the partners with somatic normal cells. PRIMA-1 is already used in phase II clinical trials related to mutated p53 cancers without apparent side effect or toxicity. We will nevertheless test PRIMA-1 for potential occular toxicity in vitro (on primary limbal cells) and in vivo (mice).


If PRIMA-1 demonstrates its ability to revert EEC limbal deficiency mouse model, as well as the production of a 3D pluristratified corneal éptihelium, the following step will be GMP-grade PRIMA-1 to be embedded into eye drops for clinical trials on EEC patients to be recruited. Moreover, our approach could become a proof of concept for many ocular diseases like aniridia. In the frame of our European network on p63, we consider to planified multicentric clinical trials in Europe.


See above

Scientific outputs and patents

The project just started


Hopital E. Herriot Lyon Banque de Tissus et Cellules HCL

INSERM Skin Research Center/hopital Saint Louis

Divers privé

ANR grant: 221 200 euros
Beginning and duration: janvier 2014 - 48 mois

Submission abstract

The p53 family of transcription factors includes p53, p63 and p73. All three family members share a high homology at the protein level but diverge in their biological functions. While p53 is a proapoptotic factor, mainly involved in protection against cancer, p63 is essential for epithelial development. Mutations on the DNA-binding domain of p63 are responsive for ectrodactyly, ectodermal dysplasia and cleft lip/palate (EEC) syndrome, a rare disease associated with progressive limbal stem cell deficiency (LSCD), eventually leading to visual impairment. Today, there is no treatment for these patients and no animal model available to screen for potential therapeutic compounds.
Recently, we reprogrammed skin fibroblasts isolated from EEC patients into induced pluripotent stem cells (EEC-iPSC) and demonstrated their inability to undergo corneal epithelial cells differentiation, as compared to control iPSC. Due to the high homology in the DNA-binding domain (DBD) of p53 and p63, we challenged PRIMA-1MET, a low molecular weight compound targeting and reactivating p53 mutants in cancer patients, during corneal commitment of EEC-iPSC. We found that PRIMA-1MET can efficiently rescue epithelial differentiation defect and re-activate p63-target gene expression of iPSC carrying mutation R304W, the most frequent mutation found in EEC patients with LSCD. Interestingly, PRIMA-1MET was much less efficient on R204W, another mutation on the DBD of p63, known to alter protein stability instead of DNA binding activity like for R304W.
Due to its ease of access and that its epithelium is avascular in its final form, cornea is a relative privileged site suitable for therapies. Successful restoration of vision in human patients is easy to monitor. Moreover, the LSCD-related visual impairment of EEC patients appears at advancing age, allowing treating patients before or at the genesis of the symptom. EEC patients with LSCD are therefore candidates of choice to evaluate PRIMA-1MET for vision correction. In a bench to bed approach, our project is designed as a pre-clinical study that aims to validate experimentally novel therapeutic tools.

The objectives of the project are:

1. to identify potential patients in France that could be treated with PRIMA-1MET, we will initiate an EEC cohort registry data of patients with LSCD.

2. to validate experimentally the ability of PRIMA-1MET to treat EEC patients. For that purpose, a two-step approach will be done. First, we will design an in vitro EEC model by reconstituting a 3D corneal epithelium with committed R304W-iPSC and R204W-iPSC, as we did on WT cells (Shalom-Feuerstein, 2012). PRIMA-1MET will be added to the culture medium during the 2 week air-liquid interface.

3. In addition, we will design an EEC-like animal model by grafting iPSC onto mouse-wounded cornea. Then, eye drops containing PRIMA-1MET will be administrated onto these mice and a regular follow up of corneal histology and vision will be performed upon time. In parallel, PRIMA-1MET will be tested in vitro and in vivo for eye cytotoxicity.

4. to revert the mutation R204W by gene therapy. We propose a gene therapy approach for EEC patients with the R204W mutation for which PRIMA-1MET is less efficient. New generation of Meganucleases called Transcription Activator-Like Effector Nucleases (TALENs) are able to efficiently induce nucleotide substitution (knock-in) on mammalian genome. We will use this unique technology from Cellectis Bioresearch to specifically revert the R304W mutation on EEC-iPSC. Then, the rev-iPSC will be committed into corneal epithelial cells to form a 3D cornea epithelium in vitro. Then, the rev-EEC cornea will be grafted into LSCD animal model to challenge their ability to restore normal vision in vivo.


ANR Programme: Programme de Recherche Translationnelle en Santé (PRTS) 2013

Project ID: ANR-13-PRTS-0001

Project coordinator:
Eric GABISON (Service d'ophtalmologie, Fondation Ophtalmologique A. de Rothschild)


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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.