摘要 : RHO is one of the most common genetic causes of autosomal dominant retinitis Pigmentosa (adRP) and there is no effective therapy for this disease. While rapidly developed CRISPR/Cas9 gene editing technology presents a promising th... 展开 RHO is one of the most common genetic causes of autosomal dominant retinitis Pigmentosa (adRP) and there is no effective therapy for this disease. While rapidly developed CRISPR/Cas9 gene editing technology presents a promising therapeutic strategy to treat adRP. A large number of studies for treating adRP using CRISPR/Cas9 have been performed based on transgenic mouse models which are affected with adRP caused by mutant mouse rhodopsin allele, the counterpart of human rhodopsin. Recently, some RHO humanized mouse models like T17M, P23H are generated, which permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems, without putting humans at risk. While available humanized mouse models are few compared to the number of known RHO mutations, but it is time-consuming and costly to build humanized mice for each mutation. We wonder whether a humanized mouse model having several mutations simultaneously can be developed, although which rarely occurs in patients, to investigate the therapeutic effect of CRISPR/Cas9 for RHO-mediated adRP in preclinical in vivo systems. Homology directed repair strategy combing with CRISPR/Cas9 was employed to introduce human RHO genomic fragment containing the replacement of mouse exon1 (mE1) after the start codon to mE5 before the stop codon and all introns by the human counterparts. The human rhodopsin could express under the control of the endogenous murine promoter both transcriptionally and translationally in vivo. Human rhodopsin in humanized mouse lines (without mutation) could replace murine rhodopsin morphologically and functionally. While human rhodopsin containing T17M, G51D, G114R, R135W and P171R mutations simultaneously in mutant humanized (Mut-Rho(wt/hum) and Mut-Rho(hum/hum)) mouse lines caused retinal degeneration. Mut-Rho(wt/hum )mice suffered from severe retinal degeneration with defective formation of rod outer segment, leaving nonrecordable electroretinogram (ERG) at 3 months. Mut- Rho(wt/hum) mice had a slower rate of photo-receptors loss. In 7-month-old Mut- Rho(wt/hum )mice, statistically reduced scotopic ERG responses were visible compared with age-matched WT mice, but the shortened outer segment and thinner outer nuclear layer could be observed from 3 months. From 7 months to 9 months, significantly abnormal scotopic ERG responses were visible and photoreceptors loss were also obvious in 9-month-old Mut-Rho(wt/hum) mice. In 12-month-old Mut- Rho(wt/hum) mice, statistically reduced scotopic and photopic ERG responses and retinal degeneration throughout the retina were visible. Because scotopic responses were more affected than photopic responses in mutant humanized mice, demonstrating that rods dysfunction was more severe than cones dysfunction and deteriorated earlier, the pattern of retinal degeneration caused by mutant human rhodopsin was a typical rod-cone decay. Immunocytochemistry in cells indicated human rhodopsin proteins with 5 mutations aggregated in the cytoplasm and were also retained in the endoplasmic reticulum. The mutant human rhodopsin also accumulated in rod inner segments and cellular bodies in vivo. In conclusion, our humanized models provide excellent opportunities to study the human rhodopsin expression patterns. Our mutant humanized heterozygotes can provide opportunities to explore gene editing therapies via CRISPR/Cas9 for these five mutations in preclinical studies, it is time-saving and cost-effective. 收起