Abstract
Purpose :
Sorsby fundus dystrophy (SFD) is an early onset macular degenerative disease that is defined by a mutation in the TIMP3 gene. Retinal pigment epithelial (RPE) cells are important in the pathogenesis of age-related macular degeneration and SFD. Cell culture models show promise in reproducing some prominent disease features. By correcting the TIMP3 mutation, we propose to determine the role of TIMP3 in disease phenotype and identify factors that might be independent of the mutation.
Methods :
Induced pluripotent stem cells (iPSCs) derived from SFD patients with the Ser204Cys TIMP3 mutation were electroporated with a ribonucleoprotein (RNP) complex of Cas9 and gRNA with donor ssDNA using Amaxa nucleofector in the presence of ROCK inhibitor and HDR enhancer. Individual colonies were selected and plated into 96-well plates. DNA was extracted and nested PCR was performed. The purified PCR product was sent for Sanger sequencing analysis and karyotype. Clones were differentiated into RPE, which were then cultured on filter membranes to determine structural and functional characteristics using TEM or IHC analysis. RPE cultured on chambered slides were stained with Alizarin Red S. Western blot was performed to determine changes in protein expression.
Results :
The Crispr/Cas9-corrected SFD iPSC-derived RPE have typical RPE cobblestone morphology and form tight junctions as observed by ZO-1 staining. Cell polarization and maturation is demonstrated by the presence of apical microvilli, basal infoldings, and tight junction formation visualized by TEM. We previously reported a significant increase in TIMP3 accumulation in the extracellular matrix (ECM) of SFD RPE. CRISPR/Cas9-corrected iPSC RPE were noted to have decreased basal TIMP3 accumulation. SFD iPSC-derived RPE form basal calcium deposits that stain for Alizarin Red S, while Crispr/Cas9-corrected SFD RPE demonstrate decreased calcium deposition (p<0.0001), similar to control levels.
Conclusions :
CRISPR/Cas9 correction of the TIMP3 mutation in SFD RPE results in reduced accumulation of extracellular TIMP3 and reversal of the formation of calcium-staining sub-RPE deposits.
This is a 2021 ARVO Annual Meeting abstract.