1. CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.
- Author
-
Burnight ER, Wiley LA, Mullin NK, Adur MK, Lang MJ, Cranston CM, Jiao C, Russell SR, Sohn EH, Han IC, Ross JW, Stone EM, Mullins RF, and Tucker BA
- Subjects
- Humans, Animals, Swine, CRISPR-Cas Systems genetics, Gene Editing, Alleles, Rhodopsin genetics, Retinitis Pigmentosa genetics, Retinitis Pigmentosa therapy
- Abstract
Rhodopsin ( RHO ) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOp CRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.
- Published
- 2023
- Full Text
- View/download PDF