Toward mechanism- and gene-based therapies for retinal degeneration

描述(申请人提供)：全世界约36,000例单纯性和家族性视网膜色素变性(RP)病例是由于杆状特异的PDE6缺陷所致，PDE6由催化(PDE6a和PDE6b)和调节(PDE6g)亚基组成。在小鼠模型中，PDE6活性低会导致RP样特征。Pde6brd1基因缺失小鼠的视网膜cGMP显著升高，视杆细胞迅速变性，在出生后3周内完成。过量的cGMP可能会引起细胞间信号的多种变化，但这些通路背后的确切机制尚不清楚。最近发现的一种弱Pde6b等位基因H620Q可以加深我们对早期变性机制的理解，并使我们能够测试新的治疗假说。与Pde6brd1小鼠一样，Pde6bH620Q突变体表现出RP样特征和视网膜cGMP显著升高。而Pde6bH620Q的降解过程明显较慢，6周后完全丧失杆状结构。此外，在出生后的前三周，Pde6bH620Q表现出相对正常的视杆组织学和可量化的视杆生理学，而Pde6brd1小鼠则不是这样。我们的长期目标是使用Pde6bH620Q小鼠找到抑制过量cGMP的细胞内效应和/或增强PDE6特异性活性的治疗方法，以防止进一步的视杆和视锥退化。目的1.建立pde6bH620Q突变小鼠体内PDE6活性低于正常水平，结合鸟苷环化酶活性正常，是否导致cGMP水平升高。目的2.在Pde6bH620Q突变体的形态退变前，鉴定cGMP的运动学(基因组中的激酶)存活和凋亡效应因子的升高。这类效应器将为延缓退变提供新的药理靶点。目的3.确定是否可以通过增加Pde6b突变棒中的PDE6活性来从基因上阻止进行性杆锥变性。我们打算使用Opsin：：Pde6b救援转基因和视网膜下注射Opsin：：Pde6b救援慢病毒来阻止变性。为了测试在杆状细胞死亡开始后，是否可以阻止杆状细胞以及逐渐的继发性锥体丢失，我们将使用三苯氧胺诱导的反向Cre/loxP系统来恢复野生型Pde6b在疾病中期的表达。公共卫生相关性：遗传性视网膜变性是无法治愈的，大约每2000人中就有一人受到影响；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，剩下的视杆和视锥感光细胞死亡能被阻止吗？我们的建议解决了一个临床相关的问题，因为当验光师/眼科医生第一次看到他们时，大多数视网膜变性患者都有严重的夜盲(视杆死亡)。如果能通过纠正疾病中期发病的主要原因来阻止视杆和视锥的进一步丢失，那么基于药物或基因的治疗方法就有希望恢复新诊断患者的日常生活能力。

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.