Project 08

CRISPR therapy in a retinal hereditary dystrophy. Translational study using an animal model and retinal organotypic cultures.

Host institution: Universidad de Alicante
PhD: Yes
Duration: 36

The use of CRISPR system, as a gene editing tool, is currently one of the strategies in which more efforts are being invested for the treatment of retinal hereditary dystrophies.

Thus, the main objective of this project is to develop a new therapeutical approach for the Central Areolar Choroidal Dystrophy mouse model using the leading technology of CRISPR.


For that, single-guide RNa for the mutated allele will be designed. Plasmids containing single-guide RNas and the high-fidelity cas9 under a cones and rods promoter derived from the human rhodopsin kinase gene will be used. First, skin fibroblast from mice Prph2 KI/WT will be obtained and maintained in in vitro cultures. After, they will be transfected with the CRISPR system using the lipofectamine method. Mutated allele will be recognized by single-guide RNa and induce a cut in this region. Non-homologous end joining system will re-join the strand adding small inserts and deletions (indels) and interrupting the mutated sequence. As the mutation of interest is dominant, its ablation by CRISPR would be enough for therapy (without need of inserting the correct sequence), maintaining the peripherin expression in the non-mutated allele. After that, the efficiency of the CRISPR-mediated therapy for the reestablishment of the wild type phenotype will be analyzed in retinal organotypic cultures from Prph2 KI/WT mice. Finally, once the efficiency of the CRISPR system has been tested in fibroblasts and retinal organotypic cultures, in vivo treatments will be done to assess the efficacy and safety of the treatment in a living organism. Functional state of the retina at different ages will be assessed by ERG and optomotor test, as described in “specific aim 2”. Retinal structure and morphology will be determined by OCT, immunohistochemistry, confocal microscopy and transmission electron microscopy.

Expected results

  1. Evaluation of the efficiency of the designed CRISPR tools in the gene editing of the mutated sequence, using Prph2 KI/WT mice fibroblasts in in vitro cultures
  2. Determination of the efficiency of CRISPR-mediated therapy for the reestablishment of the wild type phenotype in retinal organotypic cultures from Prph2 KI/WT mice
  3. Evaluation of the efficiency of CRIPR therapy to prevent retinal degeneration

Planned secondments

  1. B6, supervisor: Jan Wijnholds
    Purpose: use if AAV mediated delivery of antisense oligonucleotides
  2. B1, supervisor: Javier Sancho-Pelluz
    Purpose: characterization of oxidative stress markers in human diseased (RP)
  3. AP7, supervisor: Beatriz Llamusí
    Purpose: business training
  4. B7, supervisor: Aileen Murphy
    Purpose: economic training