Mutation-independent rhodopsin gene therapy by knockdown and replacement with a single AAV vector
用单个AAV载体敲除和替换无突变视紫红质基因的基因治疗法
许多基因增强治疗正在进入临床试验，以治疗遗传性视网膜疾病。常染色体显性遗传疾病的基因治疗面临重大障碍，其中包括等位基因异质性和沉默潜在突变基因。本文确定了一种高效的shRNA，可以结合致病基因的敲除和野生型拷贝替代的单基因治疗载体，可以在常染色体显性视网膜色素变性的犬模型中预防感光细胞死亡和视力丧失。
【摘要学习】
Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant（常染色体显性遗传） retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA 【尤其不懂这个】made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. 【没读懂这句话啊】【在一个单独的腺相关病毒(AAV)载体中，我们将该shRNA与人Rho替换cDNA相结合，使其能抵抗RNA干扰，并在野生Rho-adRP犬模型中测试了该结构。】Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.
【introduction学习】
The past two decades have seen a steep rise in the number of gene therapies entering clinical trials , and in recent years a small number of them have received marketing approval by regulatory authorities in China, Europe, and the United States.The great majority of these trials have targeted cancer, cardiovascular, and inherited monogenic diseases . Strategies for inherited monogenic diseases are by necessity based on the mechanism of disease. For the great majority of loss-of-function mutations, the strategy is gene augmentation.For mutations that cause a dominant-negative effect, gene augmentation may also provide some therapeutic benefit by diluting the deleterious effect of the mutant product【缓解负面影响】 (5, 6). However, in the case of mutations that confer a toxic gain of function, strategies that are being investigated include ablation of the gene or correction of the defect at the DNA level (e.g., CRISPR/ Cas9 gene editing), transcriptional repression, and RNA knockdown/suppression (7, 8).
Mutations in more than 250 genes are known to cause inherited retinal diseases (https://sph.uth.edu/retnet/), and considerable advances have been made in gene therapy approaches because of the accessibility of the retina. Clinical trials of gene augmentation are currently ongoing for at least six autosomal recessive diseases, three X-linked diseases, and one maternally inherited mitochondrial retinal disease (9). There are no trials for autosomal dominant retinal diseases, the most common of which is autosomal dominant retinitis pigmentosa (adRP) caused by mutations in the rhodopsin (RHO) gene (10–14). For the more than 150 identified RHO mutations, several putative （假定）pathogenic mechanisms based mostly on in vitro findings have been proposed (for review see refs. 15 and 16), but detailed characterization of RHO-adRP patient phenotype is consistent with two major categories (17–19). （经提出了几种基于体外发现推定的致病机制，RHO-adRP患者表型的详细表征与两个主要类别一致）Patients with class A mutations have severe loss of rods from early life,【视杆细胞损失】 and realistic therapeutic approaches should be directed at prolonging cone survival【延长视椎细胞的寿命】. On the other hand, patients with class B mutations can have rods that survive for decades into late adult life in some retinal regions or throughout the retina and could benefit from a gene therapy aimed at rescuing the remaining rods and preventing secondary cone cell loss (20).
主要在说前人的不足：Over the past 20 years, efforts in gene therapy for RHO-adRPhave focused on either reducing the expression of specific mutant alleles (21–28)【减少突变基因的表达】 or developing a mutation-independent strategy.The latter strategy combines knocking down the expression of both the mutant and WT RHO proteins (29–39) 【同时敲低 突变基因和WT 视紫红质基因的表达】while providing a resistant RHO cDNA that encodes for the WT protein (40–43) as a replacement. Resistance is conferred by codon modification at degenerate/wobble nucleotides within the target site, which prevents hybridization with the knockdown reagent. Such a mutation independent knockdown-and-replacement strategy aims at addressing the high allelic heterogeneity in RHO-adRP while circumventing the technical and financial challenges that would be inherent in developing multiple gene therapies for individual RHO mutations. The retinal codelivery of the two components using either two separate (42), or a single adeno-associated viral (AAV) vector (41, 43) have been explored in transgenic mice by separate research groups. However, complete prevention or arrest of the ongoing rod degeneration was not achieved.
现在工作的意义：In the present study we identified a highly effective shRNA that targets human RHO in a mutation-independent manner.When combined with a resistant form of human RHO and copackaged in a single recombinant AAV vector, this vector with dual knockdown and replacement functions provided long-term protection against retinal degeneration in a naturally occurring canine model of RHO-adRP.
【AIM】
To identified a highly effective shRNA that targets human RHO in a mutation-independent manner.
【METHODS】
1.细胞层面
293T细胞
1.筛选Rz525酶的活性
2.筛选三个shRNA（shRNA131，shRNA134和shRNA820）抑制WT和突变体（P23H，T17M）人类RHO表达的效率）
3.包装AAV，检验其纯度
2.动物层面
WT和Rho突变犬
1.视网膜下注射携带基因敲除试剂的AAV2/5载体
2.活体视网膜成像、视网膜电图、Rho蛋白和RNA定量以及视网膜组织切片形态学评价来评价Rho抑制和替代的效果
3.特殊的光照被用来加速Rho突变犬的自然病程，并快速评估视网膜下递送的AAV结构是否阻止了视网膜变性的发生
【KEY RESULTS】
1.发现（293T细胞实验进行少选）并且证实（犬实验）ShRNA820对WT&突变视紫红质的抑制作用最佳
2.开发了一种光暴露方案，通过实验触发视网膜上光感受器快速丢失，并加速自然疾病的进程。Rho突变(但不是WT)狗在急性(1分钟)光照后2周内经历中央到周边视网膜视杆细胞的完全丧失，这与临床患者在环境中遇到光强度水平相近。
【CONCLUSION】
1. identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner提出了一种新的基因治疗策略，其结合了致病基因的敲除和野生型拷贝的替换，可以防止常染色体显性视网膜色素变性犬模型中光感受器细胞的死亡和视力丧失。
2.在单个载体中组合敲除和替换，比双载体方法效率更高，安全性更好
【COMMENTS】
十分的文章也没有很难嘛.....看了等于会了.jpg
网友评论