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首页> 外文期刊>Human mutation >RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture.
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RNAi-based suppression and replacement of rds-peripherin in retinal organotypic culture.

机译：视网膜器官型培养中基于RNAi的rds-peripherin抑制和置换。

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Extensive mutational heterogeneity presents a significant barrier to the development of therapeutics for RDS-peripherin-linked autosomal-dominant retinitis pigmentosa (RP), for which more than 50 disease-related mutations have been identified to date. Mutation-independent suppression, using RNA interference (RNAi), together with simultaneous expression of a replacement rds gene (r-rds, which has been altered to escape suppression but nevertheless encodes wild-type protein) has been explored in COS-7 cells and mouse retinal explants. The efficacy of small interfering and short hairpin RNAs (si/shRNAs) silencing mouse rds, and the function of r-rds (containing degenerate substitutions in the RNAi target sequence) were analyzed at transcript (RT-PCR) and protein (ELISA) levels in COS-7 cells. "Dual-" and "triple-expression" constructs carrying the shRNA suppressor and the marker EGFP with or without the r-rds cassette were electroporated in vitro into retinal explants from 1-day-old pups. The retinae weredissociated at day 14, and transduced cells were FACS-sorted using the coexpressed EGFP marker and analyzed by RT-PCR. si/shRNAs decreased rds mRNA and protein expression by up to 82%, while r-rds was protected from suppression in COS-7 cells. Similarly, efficient RNAi-mediated suppression of endogenous rds was detected in retinal explants, while concomitant rescue of r-rds was also achieved. These data validate the concept of RNAi-based suppression coupled with replacement technology for the development of therapies targeting RDS-linked autosomal-dominant RP, and suggest that such approaches could potentially be used for other autosomal-dominant diseases with similarly extensive intragenic heterogeneity.
机译：广泛的突变异质性为RDS-外围蛋白连接的常染色体显性色素性视网膜炎（RP）疗法的发展提出了重大障碍，迄今为止已鉴定出50多种与疾病相关的突变。已经在COS-7细胞中研究了使用RNA干扰（RNAi）的突变非依赖性抑制，以及同时表达替代性rds基因（r-rds，已被改变以逃避抑制，但仍编码野生型蛋白）的表达，并且小鼠视网膜外植体。在转录本（RT-PCR）和蛋白质（ELISA）水平上分析了小干扰和短发夹RNA（si / shRNA）沉默小鼠rds的功效以及r-rds的功能（RNAi目标序列中包含简并取代）。在COS-7细胞中。将携带有shRNA抑制子和带有或不带有r-rds盒的标记EGFP的“双表达”和“三重表达”构建体体外电穿孔成1天龄幼崽的视网膜外植体。在第14天解离视网膜，并使用共表达的EGFP标记对转导的细胞进行FACS分选，并通过RT-PCR进行分析。 si / shRNA使rds mRNA和蛋白质表达降低多达82％，而r-rds在COS-7细胞中受到抑制。同样，在视网膜外植体中检测到有效的RNAi介导的内源性rds抑制，同时也实现了r-rds的同时拯救。这些数据验证了基于RNAi的抑制与替代技术相结合的概念，从而开发了针对RDS连锁的常染色体显性RP的疗法，并表明这种方法可能可用于其他具有类似广泛基因内异质性的常染色体显性疾病。

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