Accelerated maturation of ARPE‐19 cells for the translational assessment of gene therapy.

The human retinal pigment epithelium (RPE) cell line ARPE‐19 is widely used as an alternative to primary RPE despite losing many features of primary RPE. We aimed to determine whether a combination of RPE‐specific laminin (LN) and nicotinamide (NAM) could improve ARPE‐19 redifferentiation to resemble mature RPE and improve the assessment of RPE‐specific gene therapy strategies. ARPE‐19 cells were propagated on tissue culture plastic supplemented with NAM and human recombinant LN521‐coating. RPE maturation was performed by immunocytochemistry and gene expression by qPCR. Viral transduction experiments with adeno‐associated virus (AAV)1 or AAV2, carrying a VMD2‐driven GFP, were assessed at 2‐ and 4‐weeks post‐plating in the different culturing conditions with a low multiplicity of infection. The combination of LN521 coating with NAM supplementation promoted cytoskeletal and tight junction protein reorganization. The expression of maturation markers bestrophin‐1 and RPE 65 was promoted concomitantly with a reduction of several epithelial‐mesenchymal transition markers, such as TNF‐α, TGF‐β, CDH2, and vimentin. Redifferentiated ARPE‐19 transduced at low multiplicity of infection of both AAV1‐ and AAV2‐VMD2‐GFP. Expression of GFP was detected at 2 weeks and increased at 4 weeks post‐plating. AAV1 exhibited a greater expression efficacy compared to AAV2 in maturated ARPE‐19 cells already after 2 weeks with increased efficiency after 4 weeks. Our study demonstrates an improved maturation protocol for ARPE‐19 cells in vitro, mimicking an in vivo phenotype with the expression of signature genes and improved morphology. Viral‐mediated RPE‐specific gene expression demonstrates that the combination cultures mimic in vivo AAV tropism essential to test new gene therapies for RPE‐centered diseases. [ABSTRACT FROM AUTHOR]