Poster Presentation ASSCR, AGCTS, ISCT ANZ and Friends Joint Scientific Conference 2019

Gene-editing in human pluripotent stem cells for disease modelling and testing therapies in the retinal dystrophies (#203)

To Ha Loi 1 , Benjamin Nash 1 2 3 , Sharon Cunningham 4 , Samantha Ginn 4 , Anson Cheng 1 , Bruce Bennetts 2 , Ian Alexander 4 , Anai Gonzalez Cordero 5 , Robyn V Jamieson 1 3
  1. Eye Genetics Research Unit, Children's Medical Research Institute, Children's Hospital at Westmead, Save Sight Institute, University of Sydney, Sydney
  2. Sydney Genome Diagnostics, Western Sydney Genetics Program, The Children's Hospital at Westmead, Sydney Children's Hospitals Network, Sydney
  3. Discipline of Genomic Medicine, Faculty of Medicine and Health, University of Sydney, Sydney
  4. Gene Therapy Research Unit, Children's Medical Research Institute and Children's Hospital at Westmead, Sydney
  5. Stem Cell Medicine Group, Children's Medical Research Institute, Sydney

Genetic retinal dystrophies cause progressive degeneration or abnormality of photoreceptors leading to vision loss. Current genomic studies identify disease-causing variants in approximately 65% of cases, and we wish to extend the yield of this testing through analysis of gene-edited iPSCs differentiated to retinal tissues and organoids, which also provide a platform to investigate gene-based therapies. To determine the functional impact of several retinal disease variants, CRISPR/Cas9 guides were designed along with variant-specific oligonucleotides to mediate homology directed repair (HDR) in normal iPSCs, to generate isogenic lines of the novel variants under investigation. Using this approach, for a novel variant of the RPGRIP1 ciliopathy gene, we generated gene edited isogenic clonal iPSC lines with the variant correctly knocked-in to both alleles. We also identified a homozygous RPGRIP1 knockout clonal line. Retinal differentiation cultures are underway to assess changes in ciliation and function, including correct trafficking of key proteins in the photoreceptors. For gene replacement, we have modified an AAV2 vector to include an eGFP reporter and a photoreceptor-specific promoter for transgene expression in retinal organoids. Overall, we have generated isogenic iPSC lines for retinal disease modelling and gene therapy testing and will utilise this approach to examine other variants and response to therapies.

  • Have you presented your abstract at another international meeting?: No