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

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.

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The project just started

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.