Toward mechanism- and gene-based therapies for retinal degeneration

描述（由申请人提供）：全球约有36,000例单纯性和家族性视网膜色素变性（RP）是由于杆状特异性PDE6缺陷引起的，PDE6由催化（PDE6a和PDE6b）和调节（PDE6g）亚基组成。在小鼠模型中，低PDE6活性导致rp样特征。Pde6brd1缺失小鼠表现出视网膜cGMP显著升高和杆状体快速变性，并在出生后3周内完成。过量的cGMP可能会引起细胞间信号的多种变化，但这些途径的确切机制尚不清楚。最近发现的Pde6b弱等位基因H620Q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变性的时间过程明显较慢，在6周后发生完全的棒丢失。此外，Pde6bH620Q在出生后的前三周内表现出相对正常的杆状组织和可量化的杆状生理，而Pde6brd1小鼠则没有这种情况。我们的长期目标是利用Pde6bH620Q小鼠寻找抑制过量cGMP细胞内效应和/或增强PDE6特异性活性的治疗方法，以防止进一步的杆状体和锥体变性。目的1。建立如果低于正常PDE6活性，结合正常鸟苷酸环化酶活性，导致pde6bh620q突变小鼠cGMP水平升高。目标2。在Pde6bH620Q突变体出现形态退化迹象之前，确定其基因组（基因组中的激酶）存活和cGMP凋亡效应的升高。这些效应物将为延缓变性提供新的药理靶点。目标3。确定是否可以通过增加PDE6在Pde6b突变杆状体中的活性来阻止进行性杆状体变性。我们打算使用Opsin::Pde6b拯救转基因和视网膜下注射Opsin::Pde6b拯救慢病毒来阻止变性。为了测试杆状细胞死亡后杆状细胞和逐渐继发性锥体丢失是否可以被阻止，我们将采用他莫昔芬诱导的反向Cre/loxP系统在疾病中期恢复野生型Pde6b的表达。公共卫生相关性：遗传性视网膜变性是无法治愈的，每2000人中就有1人受其影响；全世界有150万人患有视网膜色素变性（RP）。一旦退化开始，剩下的杆状和锥状光感受器的死亡能停止吗？我们的建议解决了一个与临床相关的问题，因为大多数视网膜变性患者在第一次由验光师/眼科医生看到时都有明显的夜盲症（视杆死亡）。如果我们能在疾病中期通过纠正发病机制的主要原因来阻止杆状体和锥体的进一步丧失，那么就有希望通过药物或基因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.