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首页> 外文学位 >Gene Therapy Provides Long-term Visual Function in a Pre-clinical Model of Retinitis Pigmentosa.
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Gene Therapy Provides Long-term Visual Function in a Pre-clinical Model of Retinitis Pigmentosa.

机译：基因疗法在色素性视网膜炎的临床前模型中提供长期的视觉功能。

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摘要

Retinitis pigmentosa (RP) is a photoreceptor neurodegenerative disease. Patients with RP present with the loss of their peripheral visual field, and the disease will progress until there is a full loss of vision. Approximately 36,000 cases of simplex and familial RP worldwide are caused by a mutation in the rod-specific cyclic guanosine monophosphate phosphodiesterase (PDE6) complex. However, despite the need for treatment, mouse models with mutations in the alpha subunit of PDE6 have not been characterized beyond 1 month of age or used to test the pre-clinical efficacy of potential therapies for human patients with RP caused by mutations in PDE6A..;We first proposed to establish the temporal progression of retinal degeneration in a mouse model with a mutation in the alpha subunit of PDE6: the Pde6alphanmf363 mouse. Next, we developed a surgical technique to enable us to deliver therapeutic treatments into the mouse retina. We then hypothesized that increasing PDE6alpha levels in the Pde6alpha nmf363 mouse model, using an AAV2/8 gene therapy vector, could improve photoreceptor survival and retinal function when delivered before the onset of degeneration. Human RP patients typically will not visit an eye care professional until they have a loss of vision, therefore we further hypothesized that this gene therapy vector could improve photoreceptor survival and retinal function when delivered after the onset of degeneration, in a clinically relevant scenario.;For each of these studies, we used histology, autofluorescence (AF) and infrared (IR) imaging to examine the appearance of the retinal cell layers and retinal pigment epithelium (RPE) that are affected in human RP patients. We also used electroretinograms (ERGs) to measure both photoreceptor-specific and global retinal visual function in the Pde6alphanmf363 mice. For our gene therapy experiments, we utilized a vector with the cell-type-specific rhodopsin (RHO) promoter: AAV2/8(Y733F)- Rho-Pde6alpha, to transduce Pde6alphanmf363 retinas after subretinal injection at either post-natal day (P) 5 or P21. We then monitored the effects of AAV2/8(Y733F)-Rho-Pde6alpha transduction over at least a quarter of the mouse lifespan.;In the Pde6alphanmf363 mutant mouse model of RP, we found that by 2 months of age the number of photoreceptor cell nuclei is roughly halved in comparison to the 1 month time-point, and this degeneration continues until all photoreceptor cell nuclei have undergone degeneration by 4 months of age. Additionally, both loss of cone cell function and RPE atrophy are present by 5 months of age in these mice. After the development of a subretinal injection surgical procedure, we delivered the AAV2/8(Y733F)- Rho-Pde6alpha to the Pde6alphanmf363 mice at either P5 or P21. We found that a single injection enhanced survival of photoreceptors and improved retinal function. At 6 months of age, the treated eyes retained photoreceptor cell bodies, while there were no detectable photoreceptors remaining in the untreated eyes. More importantly, the treated eyes demonstrated functional visual responses even after the untreated eyes had lost all vision. Despite focal rescue of the retinal structure adjacent to the injection site, global functional rescue of the entire retina was observed. We have also determined that subretinal transduction of this rod-specific transgene at P21, when approximately half of the photoreceptor cells have undergone degeneration, has similar efficacy in rescuing cone cell function long-term as transduction before disease onset, at P5.;Therefore, we concluded that the Pde6alphanmf363 mice mimic human RP caused by mutations in PDE6A. The establishment of the temporal and biochemical characteristics of photoreceptor neurodegeneration in the Pde6alphanmf363 mice allows for future studies to test therapeutic options using this animal model, since the progression of RP can be compared to the established time-course of degeneration. Additionally, the development of a standard method for performing subretinal injections allows for comparable results after this surgical technique is used to deliver gene therapy vectors into the perinatal mouse eye. Our gene therapy studies suggest that RP due to PDE6alpha deficiency in humans, in addition to PDE6b deficiency, is also likely to be treatable by gene therapy. Furthermore, AAV2/8(Y733F)- Rho-Pde6alpha is an effective gene therapy treatment that can be utilized in the clinical setting, in human patients who have lost portions of their peripheral visual field and are in the mid-stage of disease when they first present to an eye-care professional.
机译：色素性视网膜炎（RP）是一种感光神经退行性疾病。 RP患者表现出周围视野的丧失，疾病将进展直至完全丧失视力。全世界约36,000例单纯性和家族性RP病例是由杆特异性环状鸟苷单磷酸磷酸二酯酶（PDE6）复合物的突变引起的。然而，尽管需要治疗，但PDE6的α亚基突变的小鼠模型并未超过1个月大的特征，也没有用于测试由PDE6A突变引起的RP患者的潜在疗法的临床前疗效。我们首先提出在PDE6的α亚基突变的小鼠模型中建立视网膜变性的时间进展：Pde6alphanmf363小鼠。接下来，我们开发了一种外科手术技术，使我们能够将治疗方法传递到小鼠视网膜中。然后，我们假设使用AAV2 / 8基因治疗载体在Pde6alpha nmf363小鼠模型中增加PDE6alpha的水平，可以在变性开始前交付时改善感光细胞的存活和视网膜功能。人类RP患者通常会在视力丧失之前才会去看眼科专家，因此我们进一步假设，在临床相关情况下，这种基因治疗载体在变性后开始分娩时可以改善光感受器存活和视网膜功能。对于这些研究中的每一项，我们使用组织学，自体荧光（AF）和红外（IR）成像来检查在人类RP患者中受到影响的视网膜细胞层和视网膜色素上皮（RPE）的外观。我们还使用了视网膜电图（ERG）来测量Pde6alphanmf363小鼠中的感光细胞特异性和整体视网膜视觉功能。在我们的基因疗法实验中，我们使用了带有细胞类型特异性视紫红质（RHO）启动子的载体：AAV2 / 8（Y733F）-Rho-Pde6alpha，在产后一天（P）进行视网膜下注射后转导Pde6alphanmf363视网膜5或P21。然后，我们监测了至少四分之一的小鼠寿命中AAV2 / 8（Y733F）-Rho-Pde6alpha转导的影响。在RP的Pde6alphanmf363突变小鼠模型中，我们发现到2个月大时，感光细胞的数量与1个月的时间点相比，细胞核大约减少了一半，并且这种变性一直持续到所有感光细胞核在4个月大之前都已经变性。另外，在这些小鼠中，到5个月大时，锥体细胞功能丧失和RPE萎缩都存在。在发展了视网膜下注射外科手术程序后，我们以P5或P21将AAV2 / 8（Y733F）-Rho-Pde6alpha传递给了Pde6alphanmf363小鼠。我们发现单次注射可以增强感光细胞的存活率并改善视网膜功能。在6个月大时，处理过的眼睛保留了感光细胞体，而未处理过的眼睛中没有可检测到的感光细胞。更重要的是，即使未经处理的眼睛丧失了全部视力，经过处理的眼睛也表现出功能性视觉反应。尽管对邻近注射部位的视网膜结构进行了局部抢救，但仍观察到整个视网膜的整体功能抢救。我们还确定，当大约一半的感光细胞发生变性时，P21处的这种杆特异性转基因的视网膜下转导在疾病发作前的P5时具有长期有效的挽救视锥细胞功能的类似功效;因此，我们得出的结论是，Pde6alphanmf363小鼠模拟了由PDE6A突变引起的人RP。在Pde6alphanmf363小鼠中感光神经变性的时间和生化特征的建立允许使用该动​​物模型进行未来研究以测试治疗方案，因为RP的进展可以与已确定的变性时间进行比较。另外，在使用这种外科手术技术将基因治疗载体递送到围生期小鼠眼中之后，用于进行视网膜下注射的标准方法的开发允许获得可比的结果。我们的基因疗法研究表明，除PDE6b缺乏症外，人类PDE6alpha缺乏症引起的RP也可通过基因疗法治疗。此外，AAV2 / 8（Y733F）-Rho-Pde6alpha是一种有效的基因疗法，可在临床环境中用于那些失去部分周围视野并处于疾病中期的人类患者首先送给眼保健专家。

著录项

  • 作者

    Wert, Katherine J.;

  • 作者单位

    Columbia University.;

  • 授予单位 Columbia University.;
  • 学科 Health Sciences Ophthalmology.;Health Sciences Nutrition.;Biology Neuroscience.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 158 p.
  • 总页数 158
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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