Defining Barriers to Gene Therapy

描述（由申请人提供）：在影响900万美国人的视网膜变性疾病中，视网膜色素变性（RP）是最具破坏性的。在RP和年龄相关性黄斑变性（AMD）中，视杆光感受器的进行性萎缩导致视锥光感受器的继发性死亡。基因治疗是一种潜在的手段，以加强或恢复杆的活力，从而防止二次锥损失。然而，第一个针对RP的人类基因治疗试验发现视觉功能得到改善，但并没有减缓光感受器的退化。这个基因治疗导向的建议的目标是确定治疗是否是在已经患病的视网膜的背景下实现的。由cGMP磷酸二酯酶-6（PDE 6）缺乏引起的RP是研究进行性视杆细胞自主变性和随后的非细胞自主视锥细胞丢失的理想模型。在上一个资助期间，我们成功地恢复了PDE 6活性和视网膜功能超过11个月的PDE 6缺陷小鼠通过使用病毒基因转移方法之前发生变性。我们现在提出，在目标1中，确定小鼠视杆细胞和视锥细胞是否可以在临床相关的时间窗内被拯救-也就是说，在患者通常被诊断出退行性变之后。为此，我们产生了一种新的诱导型遗传拯救系统，使我们能够有条件地逆转PDE 6缺陷，并控制表型逆转的数值，时间和空间方面。我们将使用这种非手术和强大的基因治疗方法，以确定视力恢复/保存是否受到杆拯救的时间（Aim 1a），杆拯救的数量（Aim 1a）和突变杆的非自主效应（Aim 1b）的影响。为了补充RP及其在动物模型中的拯救研究，我们在上一个资助期内开发了一种非侵入性小鼠自体荧光成像技术。由于FDA尚未接受成像生物标志物作为视网膜疾病治疗疗效的主要结局指标，因此我们将研究非侵入性成像是否可以作为评估小鼠和人类疾病进展的替代品（目的2）。在没有新的疾病预测因子来替代被认为不够敏感的常规测试的情况下，确定治疗方法的适当性和/或建立评估疗效（疾病进展或稳定）的基线是具有挑战性的。新的非侵入性成像生物标志物将使我们能够告知RP患者他们的疾病预后和治疗方案。此外，他们将解决紧迫性，建议新的准父母谁已经有一个孩子诊断为RP。综上所述，该提案肯定能够：1）定义限制介入治疗的因素; 2）验证用于药理学控制欧洲条件性小鼠诱变计划队列中任何基因的光感受器特异性表达的新工具包; 3）鉴定对于评估2期和3期临床试验中的疗效至关重要的疾病进展的新成像生物标志物; 4）帮助当前和未来的基本机制 在人类和动物模型中进行光感受器退化的研究。

DESCRIPTION (provided by applicant): Of the retinal degenerative diseases that affect 9 million Americans, retinitis pigmentosa (RP) is the most devastating. In RP and age-related macular degeneration (AMD), progressive atrophy of rod photoreceptors leads to secondary death of cone photoreceptors. Gene therapy is a potential means to strengthen or restore rod viability, thereby preventing secondary cone loss. However, the first human gene-therapy trial for RP found improved visual function but did not slow degeneration of photoreceptors. The goal of this gene therapy- oriented proposal is to determine whether therapy is achievable in the context of an already diseased retina. RP resulting from cGMP phosphodiesterase-6 (PDE6) deficiency is an ideal model for studying progressive cell- autonomous degeneration of rods and subsequent, non-cell-autonomous cone loss. During the previous funding period, we succeeded in restoring PDE6 activity and retinal function for more than 11 months in PDE6- deficient mice by using viral gene transfer methods before the onset of degeneration. We now propose, in Aim 1, to determine whether mouse rods and cones can be rescued within a clinically relevant time window - that is, after the onset of degeneration when patients are usually diagnosed. To do this, we generate a novel inducible genetic rescue system that allows us to conditionally reverse PDE6-deficiency and to control numerical, temporal and spatial aspects of phenotypic reversal. We will use this nonsurgical and robust gene therapy approach to determine if vision restoration/preservation is influenced by the timing of rod rescue (Aim 1a), the number of rods rescued (Aim 1a), and non-autonomous effects of mutant rods (Aim 1b). To complement the study of RP and its rescue in animal models, we have developed a non-invasive autofluorescence imaging technique in mice during the previous funding period. Because FDA is yet to accept imaging biomarkers as a primary outcome measure for treatment efficacy for retinal diseases, we will investigate whether non-invasive imaging can be a surrogate to assess disease progression in both mice and humans (Aim 2). Without novel disease predictors to replace conventional tests that are not deemed not sensitive enough, it is challenging to determine the appropriateness of a therapeutic approach, and/or to establish a baseline from which to assess efficacy (disease progression or stabilization). The novel non-invasive imaging biomarkers will enable us to inform RP patients their disease prognosis and treatment options. Furthermore, they will address the urgency to advise newly expectant parents who already have one child diagnosed with RP. Taken together, this proposal is certain to 1) define the factors limiting interventional therapy; 2) validate a new tool kit for pharmacological control of photoreceptor-specific expression of any gene in the European Conditional Mouse Mutagenesis Program cohort; 3) identify novel imaging biomarkers of disease progression essential for assessing efficacy in phase 2 and 3 clinical trials and 4) aid current and future basic mechanistic studies of photoreceptor degeneration in humans and animal models.