腺相关病毒载体(AAV)在视网膜色素变性基因治疗中的应用

随着分子生物学的发展 ,应用基因转移技术治疗视网膜色素变性疾病成为可能。目前研究认为腺相关病毒载体 (AAV)能使外源基因有效地导入感光细胞而且在细胞中稳定长期表达 ,而且对细胞无毒性 ,将有可能成为最有效的基因转染工具。本文对AAV病毒的形态、生长和分子生物学特征、AAV介导的视网膜细胞基因转移、AAV病毒载体在视网膜色素变性基因治疗中的应用、以及作为基因治疗载体的优缺点进行了综述     

眼部疾病的基因治疗进展

目前,全世界已有31项眼部疾病基因治疗临床试验被批准,多数仍处于研究阶段.Leber先天性黑朦(LCA)目前已开展Ⅲ期临床试验,随访时间最长6年;无脉络膜症多中心的临床试验也取得了积极效果;视网膜色素变性(RP)已开展基因治疗Ⅰ期临床试验;年龄相关性黄斑变性(AMD)基因治疗的Ⅰ期临床试验结果令人鼓舞;青光眼基因治疗中使用RNA干扰技术和优化的偶联表面活性磷脂纳米微粒也取得了良好效果.就LCA、RP、无脉络膜症、AMD和青光眼基因治疗的一些实验室及临床研究进展,包括眼部基因治疗方法、各种基因载体和常用的动物模型等进行综述.病毒载体已广泛应用于眼部疾病的基因治疗中,一些与免疫排斥和基因突变相关的潜在性风险以及个体反应的差异性促使人们去探索更安全、高效的方法.基因编辑技术的出现,必将对眼部疾病的基因治疗领域产生深远影响.     

腺相关病毒载体（AAV）在视网膜色素变性基因治疗中的应用

随着分子生物学的发展，应用基因转移技术治疗视网膜色素变性疾病成为可能，目前研究认为相关病毒载体（AAV）能使外源基因有效地导入感光细胞而且在细胞中稳定长期表达。而且对细胞无毒性，将有可能成为最有效的基因转染工具，本对AAV病毒的形态，生长和分子生物学特征，AAV介导的视网膜细胞基因转移，AAV病毒载体在视网膜色素变性基因治疗中的应用，以及作为基因治疗载体的优缺点进行了综述。     

遗传性视网膜疾病的基因研究进展

遗传性视网膜疾病是临床上最常见且危害最严重的眼科遗传性致盲疾病,主要包括各种类型的视网膜色素变性、Leber先天性黑朦、先天性静止性夜盲、卵黄样黄斑营养不良、Stargardt病等.人类基因组计划的完成及相关遗传学技术的广泛应用为遗传性视网膜疾病的基因研究提供了有效手段,目前已经取得了一系列突破性进展,特别是等位基因特异性引物延伸芯片技术的应用,极大地提高了遗传性视网膜疾病基因突变筛查的进度,到目前为止,已经鉴定出46个与遗传性视网膜疾病相关的致病基因和2497个突变位点.遗传性视网膜疾病最根本的治疗方法是基因治疗,而进行基因治疗的前提是首先要筛查到致病基因.因此有必要对国内外近年来有关遗传性视网膜疾病的基因研究近况进行综述,以供眼科同道参考.     

以腺相关病毒为载体的基因治疗在视网膜疾病中的研究进展

视网膜色素变性、Leber先天性黑蒙等视网膜变性疾病的共同特征是视网膜细胞进行性凋亡,对其目前尚缺乏有效的治疗方法.近年来的基因治疗研究为视网膜变性疾病的治疗开辟了新途径.视网膜血管性疾病目前的治疗手段如激光光凝术、外科手术以及光动力疗法等均属对症治疗,并未解决导致新生血管形成的潜在原因,基因治疗或许可填补这些治疗方式...     

腺相关病毒载体在眼科基因治疗中的应用

腺相关病毒（adeno-associated virus，AAV）属微小病毒家族，目前有8个亚型。重组AAV载体是眼科基因治疗中较为理想的载体，具有无致病性、免疫原性弱、转染细胞类型多、转染表达时间长等优点，但也存在携带目的基因较小、不容易获得较高滴度等缺点。AAV在眼科的应用途径主要是视网膜下腔注射、玻璃体腔内注射和前房内注射等局部途径应用。视网膜下腔注射可以转染光感受器细胞和视网膜色素上皮细胞，其注射方法包括经角膜前途径、巩膜自闭性隧道切口和锯齿缘后切口等途径。玻璃体腔内注射主要适用于转染视网膜神经节细胞和Mueller细胞，其注射方法主要是经巩膜途径。前房内注射主要适用于眼前节细胞的转染。目前正在研究的应用AAV治疗的眼部疾病有视网膜变性类疾病、新生血管性疾病、恶性肿瘤、角膜病及视神经保护治疗等。     

本期导读

本期以眼底疾病诊治作为重点报道内容，但也涉及白内障、青光眼、眼外伤、神经眼科等多个专业。长期以来遗传性视网膜疾病不能治疗的传统观念已被目前临床试验结果所颠覆，在述评栏目中，李文生教授等就遗传性视网膜疾病基因治疗的临床试验现状进行评论，Leber先天性黑矇目前有20余项临床试验正在招募，5项试验包括50余例已完成，结果令人鼓舞。由MERTK （RP38）基因突变引起的视网膜色素变性基因治疗临床试验在少数患者已开展，无脉络膜症基因治疗临床试验也已开展了十余例。但目前的基因治疗方法仍不能达到一劳永逸的效果，目前采用的视网膜下腔注射方法有待改进，AAV载体能承载的DNA序列长度较小以及费用昂贵等。未来应对不同的遗传性视网膜疾病以及同一疾病的不同阶段探讨基因治疗、干细胞治疗、神经保护等相结合的综合性干预措施。范志刚教授团队在专家论坛栏目中对原发性闭角型青光眼（PACG）发病机制和致病基因探索的思路进行了细致的梳理，认为解剖学上房角机械性关闭在分子机制水平可分为眼轴的发育调控、前房角相关结构的发育调控（包括晶状体和悬韧带）、视网膜-脉络膜-巩膜的发育调控。可能影响虹膜-睫状体-悬韧带-晶状体结构与发育的基因有CHAT、PLEKHA7、FEPMT2，可能影响视网膜-脉络膜-巩膜结构发育的基因有TMEM98、CRB1、BEST1，可能影响眼轴-眼球大小发育的基因有MFRP、ABCC5、PRSS56、MMP9、NOS3、HSP70，这些基因功能与PACG发生发展相关的分子机制有待进一步阐明。在分子机制角度上真性小眼球是PACG的完全外显型，而临床PACG是上述3类表型不同程度的组合，以真性小眼球为研究对象进行研究，最终有望对PACG的分子机制得到更清晰而深入地阐明，从而实现PACG基于基因型的临床亚型分类甚至干预。在论著栏目中，涉及到特发性颅内压增高症的治疗方法探讨，赵尚峰等报告了114例采用腰大池-腹腔分流术治疗随访2~16个月的效果；麻婧等报告了85眼X连锁视网膜劈裂症患者眼底蜂巢样改变的临床特征； 莫静等报告了70眼采用糖皮质激素联合免疫抑制剂治疗后仍反复发作的难治性白塞病葡萄膜炎，再联合IFNα2a皮下注射治疗随访12~36个月的效果；罗丽华等报告了60眼采用眼球侧以及联合上睑穹隆部注射曲安奈德治疗中重度活动期甲状腺相关眼病平均随访9.12个月的效果。本文还刊登了有关新疆维吾尔自治区2010-2018年白内障手术现况、白内障与人工晶状体手术技术改进、手术适应证拓展、白内障防盲项目卫生经济学评价的论著，均值得广大读者阅读学习。     

LCA2型基因治疗临床试验研究进展

Leber先天性黑矇(Leber''s congenital amaurosis,LCA)是一种遗传性致盲性眼病,在婴儿早期出现严重的视力低下或丧失丧失。该疾病的LCA2型与RPE65的突变相关。既往对于LCA2在内的遗传性视网膜疾病无有效治疗方法。近年来,随着基因治疗技术的进步,遗传性视网膜疾病的治疗进展取得了巨大进步,其中最成功的便是LCA2的基因治疗。本文简要介绍了LCA2基因治疗的发展,并对既往LCA2临床试验中的注射剂型、剂量、注射方式、测量方法、治疗效果与年龄的相关性和治疗效果的稳定性进行综述,为LCA2基因治疗进入我国临床工作提供参考及临床治疗经验。     

无脉络膜症诊疗新进展

无脉络膜症(Choroideremia ,CHM)是一种致盲的遗传性疾病,是通过X染色体长臂上的一个基因(CHM)传递的、由于编码Rab escort蛋白1(REP-1)的CHM基因的缺失或突变导致的、双眼发病的脉络膜视网膜渐进性萎缩性疾病。其特点是X染色体隐性遗传,随着视网膜光感受器、视网膜色素上皮细胞(RPE)和脉络膜毛细血管层逐渐萎缩,视力逐渐丧失。无脉络膜症的致病基因是CHM基因,位于Xq21.2,编码蛋白质REP-1。该病的男性患者发病较早,症状从早期的(十几岁～二十几岁)夜盲症逐渐发展为周边视野缺失,到晚年仅存中心管状视野,最终失明,女性携带者一般无症状。目前,无脉络膜症的诊断已有详细的诊断标准,基因治疗和视网膜移植是近年来被认为可以实现的治疗方法。其中,AAV2和AAV8的临床前研究已经完成,CHM受试者中AAV2介导的基因治疗的安全试验也已经完成。     

Leber先天性黑蒙基因研究进展

Leber先天性黑蒙(LCA)是导致婴幼儿先天性盲的严重遗传性视网膜疾病。近年发现数种与LCA相关的致病基因,主要包括GUCY2D、RPE6 5、CRX、AIPL1、RPGRIP1和CRB1,其功能涉及视网膜光电信号的传导、维生素A在视网膜的代谢、光感受器细胞的分化和形态发育、蛋白的转运和分布等。针对RPE6 5的基因治疗在动物实验中取得了一定的成果,将是未来LCA临床治疗的主要研究方向。本文就当前LCA的致病基因及其可能的发病机制、基因治疗等方面的研究进展作一综述。     

Versatility of AAV vectors for retinal gene transfer

Gene therapy represents a promising therapeutic option for many inherited and acquired retinal diseases. Recombinant adeno-associated viral vectors (AAV) are the most efficient tools to transfer genes in vivo to the retina. The recent identification of dozens of novel AAV serotypes enormously expands on the versatility of AAV as vector system for in vivo somatic gene transfer. The results from the forthcoming trials with AAV in the retina of patients with Leber Congenital Amaurosis will be critical for the rapid development of AAV-based therapeutics for retinal diseases.     

Leber先天性黑矇的基因治疗研究进展

Leber先天性黑矇(LCA)是一种严重的先天性致肓遗传性视网膜疾病.近1O年来,随着分子遗传学的发展及基因治疗技术的进步,以腺相关病毒载体介导的LCA基因治疗研究取得了令人鼓舞的进展,尤其是对LCAⅡ患者进行的RPE65基因治疗的Ⅰ期临床试验的成功使其成为眼科遗传性疾病基因治疗领域中的先行者,为今后进行其他遗传性视网膜疾病的基因治疗开辟了光明的前景.本文就目前LCA基因治疗的临床前研究及Ⅰ期临床试验的进展等方面作一综述.

Abstract：

Leber congenital amaurosis (LCA)is an early onset retinal dystrophy that causes severe visual impairment. With the development of molecular genetics and the therapeutic gene replacement technology, the adeno-associated viral (AAV) vector-mediated gene therapy for LCA achieved encouraging progress in the past decade. The success of the Phase Ⅰ clinical trials of human RPE65 gene therapy for LCA Ⅱ patients makes it a pioneer in the field of retinal gene therapy and brings light to the cure of other hereditary retinopathy. This article briefly reviews the recent developments in the preclinical animal experiments and Phase Ⅰ clinical trials for LCA.     

Molecular genetics and emerging therapies for retinitis pigmentosa: Basic research and clinical perspectives

Retinitis Pigmentosa (RP) is a hereditary retinopathy that affects about 2.5 million people worldwide. It is characterized with progressive loss of rods and cones and causes severe visual dysfunction and eventual blindness in bilateral eyes. In addition to more than 3000 genetic mutations from about 70 genes, a wide genetic overlap with other types of retinal dystrophies has been reported with RP. This diversity of genetic pathophysiology makes treatment extremely challenging. Although therapeutic attempts have been made using various pharmacologic agents (neurotrophic factors, antioxidants, and anti-apoptotic agents), most are not targeted to the fundamental cause of RP, and their clinical efficacy has not been clearly proven. Current therapies for RP in ongoing or completed clinical trials include gene therapy, cell therapy, and retinal prostheses. Gene therapy, a strategy to correct the genetic defects using viral or non-viral vectors, has the potential to achieve definitive treatment by replacing or silencing a causative gene. Among many clinical trials of gene therapy for hereditary retinal diseases, a phase 3 clinical trial of voretigene neparvovec (AAV2-hRPE65v2, Luxturna) recently showed significant efficacy for RPE65-mediated inherited retinal dystrophy including Leber congenital amaurosis and RP. It is about to be approved as the first ocular gene therapy biologic product. Despite current limitations such as limited target genes and indicated patients, modest efficacy, and the invasive administration method, development in gene editing technology and novel gene delivery carriers make gene therapy a promising therapeutic modality for RP and other hereditary retinal dystrophies in the future.     

靶向治疗视网膜疾病的研究进展

作为一种非传统但十分新颖而精准的疗法,基因手段在多种遗传性视网膜疾病的诊治中代表了一种全新而可靠的希望,尤其是先天性黑曚与Stargardt病.近年来,随着临床应用技术和诊断水平的更新及提高,利用基因手段来诊治遗传性视网膜疾病成效颇丰.本文旨在概述测序技术和基因治疗在遗传性视网膜疾病中的作用和最新的研究进展,并对目前使...     

Glaucoma progression associated with Leber’s hereditary optic neuropathy

The purpose of this article is to describe a case of open-angle glaucoma progression associated with Leber’s hereditary optic neuropathy. Single case analysis method is used. A 53-year-old woman with a previous diagnosis of glaucoma presented with progressive visual field loss. Complete ophthalmological examination and blood tests were negative for other concomitant diseases. Genetic counseling revealed mitochondrial DNA mutation compatible with the diagnosis of Leber’s hereditary optic neuropathy. In conclusion, the case describes the concomitant occurrence of open-angle glaucoma and Leber’s optic neuropathy. We hypothesize that the two diseases may have a cumulative effect on oxidative stress and retinal ganglion cell death with the consequent rapid progression of visual impairment. Screening for mitochondrial DNA mutations may be requested in patients with glaucoma who, despite pharmacologically controlled intraocular pressure, show rapid progression of the disease.     

Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy

Leber congenital amaurosis (LCA) is a rare hereditary retinal degeneration caused by mutations in more than a dozen genes. RPE65, one of these mutated genes, is highly expressed in the retinal pigment epithelium where it encodes the retinoid isomerase enzyme essential for the production of chromophore which forms the visual pigment in rod and cone photoreceptors of the retina. Congenital loss of chromophore production due to RPE65-deficiency together with progressive photoreceptor degeneration cause severe and progressive loss of vision. RPE65-associated LCA recently gained recognition outside of specialty ophthalmic circles due to early success achieved by three clinical trials of gene therapy using recombinant adeno-associated virus (AAV) vectors. The trials were built on multitude of basic, pre-clinical and clinical research defining the pathophysiology of the disease in human subjects and animal models, and demonstrating the proof-of-concept of gene (augmentation) therapy. Substantial gains in visual function of clinical trial participants provided evidence for physiologically relevant biological activity resulting from a newly introduced gene. This article reviews the current knowledge on retinal degeneration and visual dysfunction in animal models and human patients with RPE65 disease, and examines the consequences of gene therapy in terms of improvement of vision reported.     

Barriers for retinal gene therapy: separating fact from fiction

The majority of recent preclinical gene therapy studies targeting the retina have used adeno-associated virus (AAV) as the gene transfer vector. However, AAV has several limitations including the ability to generate innate inflammatory responses, the ability to cause insertional mutagenesis at a frequency of up to 56% in some tissues and a limited cloning capacity of 4.8Kb. Furthermore, AAV is known to generate limiting immune responses in humans despite the absence of similar immune responses in preclinical canine and murine studies. Three clinical trials to treat Leber's congenital amaurosis using AAV are under way. A clinical trial to treat Stargardt's using lentivirus vectors has also been recently announced. However, very limited evidence currently exists that lentivirus vectors can efficiently transduce photoreceptor cells. In contrast, very few preclinical ocular gene therapy studies have utilized adenovirus as the gene therapy vector. Nonetheless, the only two ocular gene therapy clinical trials performed to date have each used adenovirus as the vector and more significantly, in these published trials there has been no observed serious adverse event. These trials appear to be poised for Phase II/III status. Activation of cytotoxic T lymphocytes limits duration of transgene expression in the retina from first generation adenovirus vectors. However, an advanced class of adenovirus vectors referred to as Helper-dependent Adenovirus (Hd-Ad) have recently been shown to be capable of expressing transgenes in ocular tissues for more than one year. Hd-Ad vectors have many properties that potentially warrant their inclusion in the retinal gene therapy toolbox for the treatment of retinal degenerative diseases.     

提高小儿视网膜疾病的认知水平，推动我国小儿视网膜疾病研究的深入开展

小儿视网膜疾病是小儿眼科的重要组成部分。常表现为白瞳症或黄瞳症,以早产儿视网膜病变、家族性渗出性玻璃体视网膜病变、永存原始玻璃体增生症/永存胚胎血管、Coats病和视网膜母细胞瘤为代表。也可表现为眼球震颤、不追物或视功能进行性下降,以Leber先天性黑矇、Stargardt病、Best病、视锥视杆细胞营养不良等为代表。可大体分为后天获得性、遗传性和先天性发育异常三大类。随着基因检测技术和干细胞研究的进步,新药物、新设备和新技术的出现,小儿视网膜疾病的诊断治疗理念正在不断演变。我国小儿视网膜疾病的临床和科研水平正在逐步提高。     

Newer therapeutic options for inherited retinal diseases: Gene and cell replacement therapy

Inherited retinal diseases (IRD) are genotypically and phenotypically varied disorders that lead to progressive degeneration of the outer retina and the retinal pigment epithelium (RPE) eventually resulting in severe vision loss. Recent research and developments in gene therapy and cell therapy have shown therapeutic promise in these hitherto incurable diseases. In gene therapy, copies of a healthy gene are introduced into the host cells via a viral vector. Clinical trials for several genes are underway while treatment for RPE65 called voretigene neparvovec, is already approved and commercially available. Cell therapy involves the introduction of stem cells that can replace degenerated cells. These therapies are delivered to the target tissues, namely the photoreceptors (PR) and RPE via subretinal, intravitreal, or suprachoroidal delivery systems. Although there are several limitations to these therapies, they are expected to slow the disease progression and restore some visual functions. Further advances such as gene editing technologies are likely to result in more precise and personalized treatments. Currently, several IRDs such as retinitis pigmentosa, Stargardt disease, Leber congenital amaurosis, choroideremia, achromatopsia, and Usher syndrome are being evaluated for possible gene therapy or cell therapy. It is important to encourage patients to undergo gene testing and maintain a nationwide registry of IRDs. This article provides an overview of the basics of these therapies and their current status.     

Immune responses to retinal gene therapy using adeno-associated viral vectors – Implications for treatment success and safety

Recombinant adeno-associated virus (AAV) is the leading vector for gene therapy in the retina. As non-pathogenic, non-integrating, replication deficient vector, the recombinant virus efficiently transduces all key retinal cell populations. Successful testing of AAV vectors in clinical trials of inherited retinal diseases led to the recent approval of voretigene neparvovec (Luxturna) for the treatment of RPE65 mutation-associated retinal dystrophies. However, studies applying AAV-mediated retinal gene therapy independently reported intraocular inflammation and/or loss of efficacy after initial functional improvements. Both observations might be explained by targeted removal of transduced cells via anti-viral defence mechanisms. AAV has been shown to activate innate pattern recognition receptors (PRRs) such as toll-like receptor (TLR)-2 and TLR-9 resulting in the release of inflammatory cytokines and type I interferons. The vector can also induce capsid-specific and transgene-specific T cell responses and neutralizing anti-AAV antibodies which both limit the therapeutic effect. However, the target organ of retinal gene therapy, the eye, is known as an immune-privileged site. It is characterized by suppression of inflammation and promotion of immune tolerance which might prevent AAV-induced immune responses. This review evaluates AAV-related immune responses, toxicity and inflammation in studies of retinal gene therapy, identifies influencing variables of these responses and discusses potential strategies to modulate immune reactions to AAV vectors to increase the safety and efficacy of ocular gene therapy.