Defining Barriers to Gene Therapy

描述(申请人提供)：在影响900万美国人的视网膜退行性疾病中，视网膜色素变性(RP)是最具破坏性的。在RP和老年性黄斑变性(AMD)中，视杆感光细胞进行性萎缩导致视锥感光细胞继发性死亡。基因治疗是增强或恢复视杆细胞活性的一种潜在手段，从而防止继发性视锥细胞丢失。然而，首个针对RP的人类基因治疗试验发现，视觉功能得到了改善，但并没有减缓光感受器的退化。这项以基因治疗为导向的提案的目标是确定在已经患病的视网膜的情况下是否可以进行治疗。由于缺乏cGMP磷酸二酯酶-6(PDE6)而导致的RP是研究视杆细胞自主性进行性变性和随后的非细胞自主性视锥细胞丢失的理想模型。在之前的资助期间，我们通过病毒基因转移的方法成功地恢复了PDE6基因缺陷小鼠11个月以上的PDE6活性和视网膜功能，直到变性开始。我们现在建议，在目标1中，确定是否可以在临床相关的时间窗口内拯救小鼠的杆和锥--也就是在患者通常被诊断为变性之后。为了做到这一点，我们产生了一种新的可诱导的遗传救援系统，它允许我们有条件地逆转PDE6缺乏，并控制表型逆转的数字、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来确定视觉恢复/保存是否受到杆状救援的时机(目标1a)、被拯救的杆数(目标1a)和突变杆的非自主效应(目标1b)的影响。为了补充RP及其在动物模型中的拯救研究，我们在之前的资助期间开发了一种在小鼠身上的非侵入性自发荧光成像技术。由于FDA尚未接受成像生物标记物作为视网膜疾病治疗效果的主要结果衡量标准，我们将调查非侵入性成像是否可以替代评估小鼠和人类的疾病进展(目标2)。如果没有新的疾病预测指标来取代被认为不够灵敏的传统测试，就很难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。新的非侵入性成像生物标记物将使我们能够告知RP患者他们的疾病预后和治疗选择。此外，他们将解决紧急情况，建议已经有一个孩子被诊断为RP的新近怀孕的父母。综上所述，这项建议肯定会：1)确定限制介入治疗的因素；2)验证一种新的工具包，用于在欧洲条件性小鼠突变计划队列中对任何基因的光感受器特异性表达进行药理学控制；3)识别疾病进展的新的成像生物标记物，对评估2期和3期临床试验的有效性至关重要；4)有助于当前和未来的基本机制 人和动物模型中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.