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...     

腺相关病毒载体介导视网膜疾病的基因治疗研究进展

视网膜疾病分子机制研究的不断深入,促进了视网膜疾病基因治疗的进展.腺相关病毒(AAV)的毒性和免疫原性均较低,外源基因表达稳定,可以转染多种分裂期和静止期细胞,因此成为治疗视网膜疾病的有效载体.在不同的动物模型和临床试验中,已有多项研究结果证实AAV作为载体治疗视网膜疾病的安全性和有效性.目前AAV在动物疾病模型中已经进行了介导抗血管生成蛋白、神经营养因子、抗凋亡因子的表达和基因替代治疗的研究,取得了令人满意的效果.但是,AAV也存在携带外源基因能力偏小的问题,需要进一步研究并解决之.     

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。     

非病毒载体在遗传性视网膜变性基因治疗中的应用

遗传性视网膜变性(IRD)发病机制复杂多样,常常导致不可逆盲,目前尚缺乏有效的治疗方法。近年来,针对IRD的基因治疗显示出广阔的应用前景,而如何将基因药物递送至靶细胞并安全、高效地表达成为目前研究发展的关键点。病毒载体系统转染效率较高,但具有潜在的免疫反应和生物安全等问题,其临床应用的局限性使非病毒载体系统的开发应运而...     

腺相关病毒载体在眼病基因治疗中的应用及研究进展

腺相关病毒(adeno-associated virus,AAV)属微小病毒科,目前有11种血清型。AAV作为基因转移载体具有安全性好、宿主范围广、免疫原性低和携带的治疗基因长期表达等优势而成为眼病基因治疗研究的热点。影响AAV载体在眼部基因转移的因素包括AAV载体的血清型、基因转移途径、组织特异性启动子等。以AAV为载体的基因转移在视网膜疾病、青光眼、角膜病的基因治疗研究中取得了新的进展。     

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。     

重组腺相关病毒作为目标基因转移视网膜的载体的可行性研究

目的:探讨重组腺相关病毒基因(recombinant adeno-associ-ated virus gene,rAAV)载体转导绿荧光蛋白基因(green fluorescent protein,gfp)基因至视网膜的可行性。方法:18只家兔随机选取一眼玻璃体内注射rAAV-gfp,对侧眼作为对照。分别于注射后3,7,14d摘除眼球进行视网膜铺片观察视网膜的荧光。结果:家兔玻璃体注射rAAV-gfp后视网膜细胞浆内可见荧光点,提示gfp基因被有效转导至视网膜并进行荧光表达。结论:rAAV是一个可靠、简便易行的目标基因转移视网膜的载体。     

重视开展我国遗传性视网膜疾病的基因治疗研究

遗传性视网膜疾病是临床上常见且危害严重的眼科遗传性致盲疾病.我国拥有丰富的遗传性视网膜疾病人群资源,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基因研究提供了良好的平台.经过近年来的不断探索,我国对遗传性视网膜疾病的基因研究水平明显提高,部分成果已经接近国际先进水平,如常染色体显性遗传视网膜色素变性新基因的发现及功能研究,有的遗传性视网膜疾病正在从"不治之症"逐渐过渡到"可治之症"等.但就整体水平而言,不论是在遗传性视网膜疾病的基础研究还是临床研究方面与国外同类研究水平相比均存在着明显的差距.因此,我们应当抓住目前基因研究不断深入发展的机遇,集中全国眼科优势力量,创造和提供条件让更多的遗传性视网膜疾病患者尽早得到基因诊断,为早日在我国尝试进行多种遗传性视网膜疾病的基因治疗提供新的契机.     

腺伴随病毒载体介导Kringle5基因治疗早产儿视网膜病变大鼠视网膜新生血管

目的观察腺伴随病毒载体介导血管抑素Kringle5基因对SD大鼠早产儿视网膜病变（ROP）模型视网膜新生血管的影响,探寻治疗ROP的新方法。方法亚克隆构建pSNAV-Kringle5-gfp载体，腺伴随病毒包装形成rAAV-Kringle5-gfp。21只鼠高氧环境下建立ROP模型, 随机分为实验组和对照组（各21只眼）。两组各取18只眼作视网膜组织切片；各剩3只眼作聚合酶链反应（RT-PCR）和Western blotting检测。另设正常对照5只大鼠。实验组每只眼注入滴度为2.5×10¹²vg/ml的rAAV-Kringle5-gfp 10 μl，对照组每只眼注入滴度为2.5×10¹¹vg/ml的rAAVLacZ 10 μl。荧光显微镜下观察目的基因在眼组织中的表达。12周后,麻醉下处死大鼠，行血管内皮细胞Ⅷ因子相关抗原染色、血管内皮细胞细胞核记数。结果pSNAV-Kringle5-gfp载体经测序正确;腺伴随病毒载体介导血管抑素Kringle5 在玻璃体腔及视网膜有大量表达; 目的基因在mRNA水平及蛋白水平均有表达;实验组、阴性对照组视网膜表面内皮细胞细胞核分别为（19.954 2±3.825 7）、（7.335 2±2.731 3）个，两组比较差异有统计学意义（P＜0.01）。结论腺伴随病毒载体介导血管抑素Kringle5对ROP视网膜新生血管具有抑制作用。(中华眼底病杂志,2005,21:288-291)     

Gene therapy in inherited retinal degenerative diseases,a review

Hereditary diseases of the retina represent a group of diseases with several heterogeneous mutations that have the common end result of progressive photoreceptor death leading to blindness. Retinal degenerations encompass multifactorial diseases such as age-related macular degeneration, Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa. Although there is currently no cure for degenerative retinal diseases, ophthalmology has been at the forefront of the development of gene therapy, which offers hope for the treatment of these conditions. This article will explore an overview of the clinical trials of gene supplementation therapy for retinal diseases that are underway or planned for the near future.     

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.     

Vector platforms for gene therapy of inherited retinopathies

Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina's compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs' limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations.     

Early and late stage gene therapy interventions for inherited retinal degenerations

Inherited and age-related retinal degeneration is the hallmark of a large group of heterogeneous diseases and is the main cause of untreatable blindness today. Genetic factors play a major pathogenic role in retinal degenerations for both monogenic diseases (such as retinitis pigmentosa) and complex diseases with established genetic risk factors (such as age-related macular degeneration). Progress in genotyping techniques and back of the eye imaging are completing our understanding of these diseases and their manifestations in patient populations suffering from retinal degenerations. It is clear that whatever the genetic cause, the majority of vision loss in retinal diseases results from the loss of photoreceptor function. The timing and circumstances surrounding the loss of photoreceptor function determine the adequate therapeutic approach to use for each patient. Among such approaches, gene therapy is rapidly becoming a therapeutic reality applicable in the clinic. This massive move from laboratory work towards clinical application has been propelled by the advances in our understanding of disease genetics and mechanisms, gene delivery vectors, gene editing systems, and compensatory strategies for loss of photoreceptor function. Here, we provide an overview of existing modalities of retinal gene therapy and their relevance based on the needs of patient populations suffering from inherited retinal degenerations.     

慢病毒载体在眼科基因治疗的应用

随着分子生物学技术的进步和基因遗传学的发展,慢病毒载体系统不断地得到改良及优化,并以其特有的优势,逐渐成为眼科基因治疗的良好载体之一,由于其来源特殊,其生物安全性也备受关注。目前慢病毒已应用于多种眼科疾病的基因治疗研究中,并取得较好成果,为许多疾病的治疗带来了希望,但是我们要将其真正应用于临床,还任重而道远。     

视网膜相关疾病基因治疗的现状

随着对各种疾病认识的深入和医疗手段的提高,精准医学要求我们将研究细化到分子水平。基因治疗以其独有的优势成为遗传性疾病的重要治疗手段。视网膜由于其特有的优势一直处在基因治疗研究的最前沿,已在体外实验和疾病模型中获得了较为成熟的理论和技术,已有部分转化为临床成果。本文重点综述视网膜相关疾病的基因治疗现状,分析其研究成果、突变位点和疾病模型,希望对后续疾病的研究方向和治疗手段提供有价值的参考。我们还列出了目前常用的基因治疗载体,以供相关研究者选择和改进。     

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.