Toward mechanism- and gene-based therapies for retinal degeneration

描述（由申请人提供）：全世界约有36，000例单纯性和家族性视网膜色素变性（RP）是由于视杆细胞特异性PDE 6缺陷所致，PDE 6由催化（PDE 6a和PDE 6 b）和调节（PDE 6 g）亚基组成。在小鼠模型中，低PDE 6活性导致RP样特征。Pde 6 brd 1敲除小鼠表现出视网膜cGMP的显著升高和快速的视杆变性，其在出生后3周内完成。过量cGMP诱导的细胞间信号传导可能存在多种变化，但这些途径的确切机制尚不清楚。最近发现的弱Pde 6 b等位基因H620 Q可以进一步了解早期变性机制，并使我们能够测试新的治疗假设。与Pde 6 brd 1小鼠一样，Pde 6 bH 620 Q突变体显示RP样特征和视网膜cGMP的显著升高。然而，Pde 6 bH 620 Q的退化的时间过程明显较慢，在6周后发生完全的杆损失。此外，对于出生后的前三周，Pde 6 bH 620 Q显示出相对正常的视杆组织学和可量化的视杆生理学，这在Pde 6 brd 1小鼠中并非如此。我们的长期目标是使用Pde 6 bH 620 Q小鼠找到抑制过度cGMP的细胞内效应和/或增强PDE 6比活性的疗法，以防止进一步的视杆细胞和视锥细胞变性。目标1。确定如果低于正常的PDE 6活性，结合正常的鸟苷酸环化酶活性，会导致Pde 6 bH 620 Q突变小鼠中cGMP水平升高。目标2.在变性的形态学迹象之前，鉴定Pde 6 bH 620 Q突变体中cGMP升高的kinomic（基因组中的激酶）存活和凋亡效应物。这种效应物将提供新的药理学靶点以延缓变性。目标3：确定进行性视杆细胞-视锥细胞变性是否可以通过增加PDE 6 b突变视杆细胞中的PDE 6活性而在遗传上被阻止。我们打算使用视蛋白：：Pde 6 b拯救转基因和视网膜下注射视蛋白：：Pde 6 b拯救慢病毒来阻止变性。为了测试在杆死亡开始后是否可以阻止杆以及逐渐的次级视锥损失，我们将采用他莫昔芬诱导的反向Cre/loxP系统来恢复疾病中期表达的野生型Pde 6 b。公共卫生相关性：视网膜变性的遗传形式是不可治愈的，并且影响大约2000人中的一个;全世界有150万人受到视网膜色素变性（RP）的影响。一旦退化开始，剩余的视杆细胞和视锥细胞的死亡是否可以停止？我们的建议解决了一个临床相关的问题，因为大多数视网膜变性患者在第一次被验光师/眼科医生看到时都有明显的夜盲症（杆死亡）。如果人们可以通过在疾病中期纠正发病机制的主要原因来阻止进一步的视杆细胞和视锥细胞损失，那么就有希望通过基于药物或基因的治疗来恢复新诊断患者的日常生活活动。

DESCRIPTION (provided by applicant): About 36,000 cases of simplex and familial retinitis pigmentosa (RP) worldwide are due to defects in rod-specific PDE6, which consists of catalytic (PDE6a and PDE6b) and regulatory (PDE6g) subunits. In mouse models, low PDE6 activity leads to RP-like features. The Pde6brd1 null mouse exhibits dramatic elevation of retinal cGMP and rapid rod degeneration, which is complete within 3 weeks after birth. There are likely multiple changes in intercellular signaling induced by excessive cGMP, but the exact mechanisms underlying these pathways are unknown. A recently discovered weak Pde6b allele, H620Q, can further our understanding of early degeneration mechanisms and enable us to test novel therapeutic hypotheses. Like Pde6brd1 mice, Pde6bH620Q mutants show RP-like features and dramatic elevation of retinal cGMP. However, the time course of degeneration of Pde6bH620Q is significantly slower, with complete rod loss occurring after 6 weeks. Moreover, for the first three weeks after birth Pde6bH620Q display relatively normal rod histology and quantifiable rod physiology, which is not the case in Pde6brd1 mice. Our long-term goal is to use Pde6bH620Q mice to find therapies that inhibit the intracellular effects of excessive cGMP and/or enhance PDE6 specific activity to prevent further rod and cone degeneration. Aim 1. Establish if lower than normal PDE6 activity, combined with normal guanylate cyclase activity, results in elevated cGMP levels in Pde6bH620Qmutant mouse. Aim 2. Identify kinomic (kinases in the genome) survival and apoptotic effectors of cGMP rise in Pde6bH620Q mutants before morphological signs of degeneration. Such effectors will provide novel pharmacological targets to retard degeneration. Aim 3. Determine if progressive rod-cone degeneration can be genetically arrested by increasing PDE6 activity in Pde6b mutant rods. We intent to halt degeneration using Opsin::Pde6b rescue transgene and subretinal injections of Opsin::Pde6b rescue lentivirus. To test if the rod as well as gradual secondary cone loss can be arrested after the onset of rod death, we will employ a tamoxifen-inducible reverse Cre/loxP system to restore wild-type Pde6b in expression in mid-phase of the disease. PUBLIC HEALTH RELEVANCE: Inherited forms of retinal degeneration are incurable and affect about one in 2000 people; 1.5 million people worldwide are affected by retinitis pigmentosa (RP). Can the remaining rod and cone photoreceptor death be halted once degeneration has begun? Our proposal addresses a clinically relevant question, as most retinal degeneration patients have significant night blindness (rod death) when they are first seen by an optometrist/ophthalmologist. If one can impede further rod and cone loss by correcting the primary cause of the pathogenesis at the mid-stage of disease, then there is hope for a drug- or gene-based therapy to restore activities of daily living for newly diagnosed patients.