Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.
Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.
复制标题
RPE65的慢病毒基因转移在Leber先天性amurosis小鼠模型中挽救了锥体的生存和功能。
DOI：
10.1371/journal.pmed.0030347
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发表时间：
2006-10
期刊：
影响因子：
15.8
通讯作者：
Arsenijevic, Yvan
中科院分区：
文献类型：
--
作者：
Bemelmans, Alexis-Pierre;Kostic, Corinne;Crippa, Sylvain V.;Hauswirth, William W.;Lem, Janis;Munier, Francis L.;Seeliger, Mathias W.;Wenzel, Andreas;Arsenijevic, Yvan
RPE 65在视网膜色素上皮中特异性表达，并且对于视杆和视锥视蛋白的发色团11-顺式-视网膜的再循环是必需的。在人类中，RPE 65的突变导致Leber先天性黑蒙或早发性视网膜营养不良，一种严重的视网膜色素变性。RPE 65缺陷的基因治疗的可行性的证据已经在Leber先天性黑蒙的狗模型中建立，但是对锥体功能的拯救，尽管对人类高敏锐度视力至关重要，但从未被严格证明。在Rpe 65基因敲除小鼠中，光感受器显示出急剧降低的光敏感性，并且容易退化，视锥光感受器在疾病的早期阶段丧失。在本研究中，我们解决的问题，是否应用慢病毒载体表达Rpe 65小鼠cDNA防止视锥细胞变性和恢复视锥细胞功能Rpe 65敲除小鼠。在Rpe 65缺陷小鼠中视网膜下注射载体导致Rpe 65在视网膜色素上皮中的持续表达。视网膜电图记录显示Rpe 65基因转移使视网膜功能恢复到接近正常的模式。我们使用视锥细胞特异性标记物进行了组织学分析，并证明Rpe 65基因转移完全防止视锥细胞变性，直到至少4个月，在这个年龄，几乎所有的视锥细胞在未经处理的Rpe 65缺陷小鼠中已经退化。我们建立了一个算法，允许预测的锥拯救区作为转基因表达的函数，这应该是一个有用的工具，为未来的临床试验。最后，在RPE 65和视杆细胞转导素都缺乏的小鼠中，当在疾病的早期阶段应用时，Rpe 65基因转移恢复了视锥细胞功能。通过证明慢病毒介导的Rpe 65基因转移可以保护和恢复Rpe 65-/-小鼠的视锥细胞功能，这项研究强化了基因治疗对RPE 65缺陷的治疗价值，提出了视网膜视锥细胞保留治疗方法，并评估了潜在的有效治疗方法。为此目的的病毒载体。在Rpe 65-/-小鼠Leber先天性黑蒙模型中，注射表达Rpe 65小鼠cDNA的慢病毒载体能够防止视锥变性并恢复视锥功能。利伯先天性黑蒙（LCA）是一组导致婴儿和儿童失明的遗传性疾病的名称。许多不同基因中的任何一个的变化都可能导致失明，这会影响出生后或出生后不久的视力。这种情况最早是由德国医生西奥多·勒伯在19世纪描述的，因此名字的第一部分;“黑蒙”是失明的另一个词。一种称为视网膜色素上皮特异性蛋白65 kDa（RPE 65）的基因突变导致了约10%的LCA病例。RPE 65之所以这么叫是因为它在色素上皮细胞中表达，色素上皮细胞是与光敏细胞相邻的细胞层，大小为65千道尔顿。这种基因的产物对于一种称为11-顺式-视网膜的物质的再循环是必不可少的，这种物质对于视网膜的感光杆和视锥细胞捕捉光线是必需的。如果该基因异常，视网膜对光的敏感性就会急剧降低，但也会导致感光细胞本身受损。像这样的眼病可能通过基因疗法来治疗，基因疗法的工作原理是用正常功能的基因替换有缺陷的基因，通常是将正常基因的一个拷贝放入无害的病毒中，然后将其注射到受影响的组织中，在这种情况下，就是眼睛。这里的研究人员想看看是否表达野生型RPE 65使用一种特殊类型的基因载体，可以携带大片段的DNA转录-慢病毒载体-可以防止视锥细胞的退化和恢复视锥细胞功能，在这种类型的LCA小鼠模型中，Rpe 65基因已经被基因删除。将正常基因注射到Rpe 65缺陷小鼠的视网膜中，导致视网膜色素上皮细胞中蛋白Rpe 65的持续表达。对这些小鼠眼睛活动的电子记录显示，Rpe 65基因转移使视网膜功能恢复到接近正常的水平。此外，Rpe 65基因转移完全阻止了视锥细胞变性，直到至少四个月，在这个年龄，几乎所有的视锥细胞都在未经治疗的Rpe 65缺陷小鼠中退化。这些发现表明，理论上可以通过基因治疗来治疗这种类型的失明。然而，由于这项研究是在小鼠中进行的，因此在明确该治疗是否适用于人类之前，还需要采取许多其他步骤。这些步骤包括证明该病毒在人类中是安全的，并进行实验以确定需要多少剂量的病毒以及治疗效果将持续多长时间。另一个问题是，是否有必要（甚至可能）在儿童早期或儿童开始失去视力时治疗受影响的儿童。请通过http://dx.doi.org/10.1371/journal.pmed.0030347上的本摘要在线版本访问这些网站。 视网膜研究基金会有关于利伯氏先天性黑蒙的详细信息联系一个家庭是一个英国组织，旨在使患病儿童的家庭相互联系。包括那些致力于治疗眼疾的人。该基金会提供有关导致失明的疾病的信息，包括利伯氏先天性黑蒙症。
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65 −/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose. In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function. Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves. Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed. Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice. These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030347. The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
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DOI：
10.1073/pnas.250478897
发表时间：
2000-12-05
影响因子：
11.1
作者：
Calvert, PD;Krasnoperova, NV;Lem, J
通讯作者：
Lem, J
影响因子：
4.4
作者：
Chen, Y;Moiseyev, Q;Ma, JX
通讯作者：
Ma, JX
影响因子：
1.8
作者：
Ekesten, B;Gouras, P;Salchow, DJ
通讯作者：
Salchow, DJ
影响因子：
1.4
作者：
Narfström, K;Vaegan;Seeliger, M
通讯作者：
Seeliger, M
DOI：
10.1073/pnas.94.13.6916
发表时间：
1997-06-24
影响因子：
11.1
作者：
Flannery, JG;Zolotukhin, S;Hauswirth, WW
通讯作者：
Hauswirth, WW