基因编辑治疗视网膜疾病的研究进展

成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关核酸酶（Cas）系统和诱导Cas蛋白到达基因组预定区域的RNA基因编辑技术,在视网膜疾病的治疗中具有广大应用前景。迄今为止,已知有200多个基因可通过干扰视锥细胞、视杆细胞或视网膜色素上皮细胞的发育、功能和存活,从而导致遗传性视网膜营养不良（IRD）。CRISPR/Cas系统可以永久性、精确地替代或消除致病基因突变,治疗包括视网膜色素变性和Leber先天性黑矇等多种IRD。不仅如此,多因素相关的年龄相关性黄斑病变也被证实可通过CRISPR/Cas系统得到治疗缓解。本文主要论述CRISPR /Cas系统的作用机制以及该项基因编辑技术在视网膜疾病中的应用前景。     

CRISPR/Cas9系统在晶状体相关疾病发病机制研究中的应用

成簇规律间隔短回文重复序列系统(clustered regularly interspaced short palindromic repeats,CRISPR)/以及CRISPR相关蛋白9(Cas9)系统是一种新型的基因组编辑技术,具有基因敲除效率高、精准定位、易于操作、实验周期短、成本低廉等优点。CRISPR/Cas9系统的出现为眼病尤其是晶状体相关疾病的诊治提供了新的思路,在先天性白内障发病机制的研究及治疗、先天性白内障模型的构建、晶状体相关性疾病突变基因位点的确定、后发性白内障防治等方面均有一定的作用。     

CRISPR/Cas系统在人眼相关疾病中的应用

CRISPR/Cas系统原是在细菌的免疫系统内发现的用来抵抗外源遗传物质(如噬菌体病毒)的一种防御系统。由于其精确的靶向功能,CRISPR/Cas系统被开发成一种高效的基因编辑工具,被广泛地应用于生命科学研究的各个领域,并取得了革命性的进展。本文回顾性研究相关文献中关于CRISPR/Cas系统在人眼相关疾病中的应用,并进行总结。     

微小RNA在视网膜中的表达及其与视网膜疾病的关系

微小RNA(microRNA.miRNAs)是一类调控基因表达的非编码小RNA.miRNA的表达具有组织特异性,并参与细胞生长、发育、分化、增生以及凋亡等过程.近年来,人们也发现miRNA在视网膜不同细胞中呈特异表达,尽管尚无直接证据,但这些差异表达的miRNA可能与视网膜疾病如视网膜色素变性以及糖尿病视网膜病变的发生发展有着密切的联系.本文对miRNA在视网膜中的特异表达以及miRNA在视网膜疾病中的作用等研究现状进行总结,同时对miRNA在视网膜相关疾病的治疗方面的前景进行探讨.     

PAX6基因与视网膜疾病关系的研究进展

PAX6基因在胚胎发育过程中起重要作用,PAX6基因突变有可能导致先天性无虹膜、视网膜母细胞瘤、黄斑发育不良、Peters异常等眼病。本文主要就PAX6基因的基本背景知识以及PAX6与视网膜疾病的关系进行综述。     

视网膜变性Pde6b基因突变小鼠治疗的研究进展

视网膜变性动物模型是研究人类遗传性视网膜变性疾病治疗的重要工具,目前常用于基础研究的视网膜变性动物模型包括系列视杆细胞变性小鼠(rd),如rd1小鼠、rd10小鼠和nmf137小鼠,这些模型鼠均为磷酸二酯酶β亚基(Pde6b,PDEβ)基因突变的小鼠模型,因其发病机制与人类具有某些同源性,因而已大量应用于视网膜变性疾病的基因诊断和治疗研究中.为了探索更为有效的治疗方法,就3种模型鼠的发病机制、药物治疗、神经营养治疗以及干细胞治疗方法、疗效等的研究进展进行综述.     

视网膜变性磷酸二酯酶及其编码基因的研究

环鸟苷酸磷酸二酯酶是感受器进行光传导过程中的关键成分，也是近几年研究视网膜变性疾病中最引人注目的课题之一。本文从环鸟苷酸磷酸二酯酶在在光传导过程中的生理机制，ｒｄ小鼠视网膜的生化分析及基因突变，转基因ｒｄ／ｒｄ小鼠，环鸟苷酸磷酸二酯酶的编码基因及其在组织细胞中的表达，调控，用于检测视网膜色素变性患者的基因突变等方面，较全面地总结了近几年对视网膜环鸟苷酸磷酸二酯酶的研究进展。     

视网膜变性疾病的动物模型和治疗研究进展

1　视网膜变性疾病的动物模型1.1　自发视网膜变性动物模型视网膜细胞的分化和发育的基本机制及视觉传导的光电转化反应从无脊椎动物到脊椎动物都很相似,所涉及的基因和蛋白在不同种类的动物,低到果蝇高到人类,大多高度保守。许多不同种的动物患有与人相似的自然发生的视网膜变性,因而为研究人类视网膜变性疾病的病因及其病理机制提供了天然的模型。例如发生在RCS大鼠、Rd(retinaldegeneration)鼠及Rds(retinaldegenerationslow)鼠中的视网膜变性作为模型,对研究人类视网膜变性的分子遗传缺陷、生物化学缺陷的组织病理学改变已作出了很大…     

视网膜色素变性疾病相关前体mRNA剪接因子研究进展

视网膜色素变性(RP)是临床上常见的一组遗传性视网膜变性疾病,并具有显著的遗传异质性.前体mRNA(pre-mRNA)的剪接是指在剪接体的催化作用下,将基因初始mRNA中的内含子去掉并将外显子拼接的过程.在目前已发现的80多个RP致病基因中,有8个(PRPF3、PRPF8、PRPF31、PRPF6、PRPF4、SNRNP200、RP9和DHX38)在全身广泛表达并与前体mRNA剪接相关,然而这些基因突变只引起眼部病变的机制尚不清楚.本文介绍了pre-mRNA的剪接过程,总结了与RP相关的pre-mRNA的基因突变和8种剪接基因在剪接过程中的作用,并探讨相关基因突变仅引起眼部病变的机制.     

视网膜发育中的神经干细胞

视网膜作为脑组织的延伸,存在大量神经干细胞,在内外源性因子机制及各基因调节机制作用下,增殖分化为视网膜各型神经元及神经胶质细胞。应用无血清培养及单细胞克隆技术可分离培养出特异性表达nestin的视网膜干细胞。由于视网膜于细胞具备自我更新能力及多分化潜能,有望用于退行性神经疾病如:视网膜色素变性、老年黄斑变性、晚期青光眼等的细胞替代治疗或药物、基因治疗的载体。     

Applications of CRISPR/Cas9 in retinal degenerative diseases

Gene therapy is a potentially effective treatment for retinal degenerative diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed as a new genome-editing tool in ophthalmic studies. Recent advances in researches showed that CRISPR/Cas9 has been applied in generating animal models as well as gene therapy in vivo of retinitis pigmentosa (RP) and leber congenital amaurosis (LCA). It has also been shown as a potential attempt for clinic by combining with other technologies such as adeno-associated virus (AAV) and induced pluripotent stem cells (iPSCs). In this review, we highlight the main points of further prospect of using CRISPR/Cas9 in targeting retinal degeneration. We also emphasize the potential applications of this technique in treating retinal degenerative diseases.     

CRISPR-Cas9基因编辑技术在眼科疾病中的应用

规律成簇间隔短回文重复（clustered regularly interspaced short palindromic repeat,CRISPR）及相关核酸内切酶9（CRISPR associated protein,Cas9）技术是一种由RNA指导核酸内切酶的基因编辑技术。该技术以其操作简便、基因敲除效率高、靶向精准、周期短等特点迅速被用于多个物种的基因组编辑及疾病基因治疗中。本文旨在对CRISPR-Cas9技术在构建眼科疾病模型和治疗眼科疾病中的应用进展进行综述。     

Personalised genome editing – The future for corneal dystrophies

The potential of personalised genome editing reaching the clinic has come to light due to advancements in the field of gene editing, namely the development of CRISPR/Cas9. The different mechanisms of repair used to resolve the double strand breaks (DSBs) mediated by Cas9 allow targeting of a wide range of disease causing mutations. Collectively, the corneal dystrophies offer an ideal platform for personalised genome editing; the majority of corneal dystrophies are monogenic, highly penetrant diseases with a known pattern of inheritance. This genetic background coupled with the accessibility, ease of visualisation and immune privilege status of the cornea make a gene editing strategy for the treatment of corneal dystrophies an attractive option. Off-target cleavage is a major concern for the therapeutic use of CRISPR/Cas9, thus current efforts in the gene editing field are focused on improving the genome-wide specificity of Cas9 to minimise the risk of off-target events. In addition, the delivery of CRISPR/Cas9 to different tissues is a key focus; various viral and non-viral platforms are being explored to develop a vehicle that is highly efficient, specific and non-toxic. The rapid pace and enthusiasm with which CRISPR/Cas9 has taken over biomedical research has ensured the personalised medicine revolution has been realised. CRISPR/Cas9 has recently been utilised in the first wave of clinical trials, and the potential for a genome editing therapy to treat corneal dystrophies looks promising. This review will discuss the current status of therapeutic gene editing in relation to the corneal dystrophies.     

基因组编辑技术在遗传性视网膜营养不良基因治疗和细胞治疗中的应用

遗传性视网膜营养不良（inherited retinal dystrophies,IRDs）是发展中国家青年人群视力障碍的主要原因,这些疾病呈现不可逆的视觉功能障碍和（或）神经视网膜细胞的丢失,显著影响患者生活质量。由于眼睛的解剖学可及性和免疫赦免特性,眼科研究一直处于创新的基因治疗和细胞治疗的前沿,这两种疗法均具有作为IRDs患者治疗方法的巨大潜力。群集规则间隔短回文重复序列(CRISPR)和CRISP相关蛋白(Cas)等基因组编辑技术为编辑人类基因组提供了准确和有效的方法,成为治疗IRDs的一个极具前景的替代方案。目前,基因组编辑和干细胞技术领域迅猛发展,可以联合为患者提供精确和个性化的治疗,但尚存在缺陷和不足,需进一步研究。     

RNA-targeting strategies as a platform for ocular gene therapy

Genetic medicine is offering hope as new therapies are emerging for many previously untreatable diseases. The eye is at the forefront of these advances, as exemplified by the approval of Luxturna® by the United States Food and Drug Administration (US FDA) in 2017 for the treatment of one form of Leber Congenital Amaurosis (LCA), an inherited blindness. Luxturna® was also the first in vivo human gene therapy to gain US FDA approval. Numerous gene therapy clinical trials are ongoing for other eye diseases, and novel delivery systems, discovery of new drug targets and emerging technologies are currently driving the field forward. Targeting RNA, in particular, is an attractive therapeutic strategy for genetic disease that may have safety advantages over alternative approaches by avoiding permanent changes in the genome. In this regard, antisense oligonucleotides (ASO) and RNA interference (RNAi) are the currently popular strategies for developing RNA-targeted therapeutics. Enthusiasm has been further fuelled by the emergence of clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated (Cas) systems that allow targeted manipulation of nucleic acids. RNA-targeting CRISPR-Cas systems now provide a novel way to develop RNA-targeted therapeutics and may provide superior efficiency and specificity to existing technologies. In addition, RNA base editing technologies using CRISPR-Cas and other modalities also enable precise alteration of single nucleotides. In this review, we showcase advances made by RNA-targeting systems for ocular disease, discuss applications of ASO and RNAi technologies, highlight emerging CRISPR-Cas systems and consider the implications of RNA-targeting therapeutics in the development of future drugs to treat eye disease.     

Using CRISPR Interference as a Therapeutic Approach to Treat TGFβ2-Induced Ocular Hypertension and Glaucoma

PurposePrimary open angle glaucoma (POAG) is a leading cause of blindness worldwide with elevated intraocular pressure (IOP) as the most important risk factor. POAG IOP elevation is due to pathological changes in the trabecular meshwork (TM). Elevated TGFβ2 contributes to these changes and increases IOP. We have shown that histone hyperacetylation is associated with TGFβ2 elevation in the TM. In this study, we determined if clustered regularly interspaced short palindromic repeats (CRISPR) interference could specifically deacetylate histones and decrease TGFβ2 in the TM.MethodsWe tested the efficiency of different promoters in driving KRAB-dCAS9 expression in human TM cells. We also screened and determined the optimal sgRNA sequence in the inhibition of TGFβ2. Chromatin immunoprecipitation-qPCR was used to determine the binding of KRAB-dCAS9. An adenovirus-mediated TGFβ2-induced ocular hypertension (OHT) mouse model was used to determine the effect of the CRISPR interference system in vivo.ResultsWe found that the CRISPR interference system inhibited TGFβ2 expression in human TM cells, and properly designed sgRNA targeted the promoter of the TGFβ2 gene. Using sgRNA targeting the CMV promoter of the Ad5-CMV-TGFβ2 viral vector, we found that lentivirus-mediated KRAB-dCAS9 and sgRNA expression was able to inhibit Ad5-CMV-TGFβ2-induced OHT in C57BL/6J female and male mice eyes. This inhibition of OHT was associated with decreased levels of TGFβ2 and extracellular matrix proteins in the mouse eye.ConclusionsOur results indicate that CRISPR interference is a useful tool for gene inhibition and may be a therapeutic approach to treat TGFβ2-induced OHT.     

Usher综合征与USH2A基因的研究进展

Usher综合征(USH)是一种以先天性感音神经性聋和视觉功能进行性丧失为特征的遗传性疾病,具有高度的遗传异质性及临床异质性,目前尚无有效的预防和治愈方法。目前已知USH有14个致病基因,USH2A突变是其最常见的原因。随着对USH2A基因研究的深入,USH2A致病机制、动物模型建立、临床诊断以及基于基因治疗、细胞移植和RNA剪接的治疗等方面研究皆取得了巨大进展。如,USH2A的突变导致参与外周纤毛区运输功能的USH复合体蛋白产生缺陷;基于此致病机制的小鼠及斑马鱼动物模型被建立,但存在其各自局限性;通过成簇规律间隔的短回文重复序列及其相关蛋白9系统对患者来源诱导多功能干细胞进行纠正后,将其诱导为视器官以进行临床的功能纠正性移植和基于反义寡核苷酸的RNA剪接治疗在此病的治疗中属于前景性研究。