Defining Barriers to Gene Therapy

项目总结 在影响900万美国人的视网膜退行性疾病中，视杆细胞和视锥细胞感光细胞营养不良 可以说是最具破坏性的。基因治疗是一种潜在的增强光感受器活性的手段。 然而，首个针对视网膜变性的人类基因治疗试验发现，视觉功能有所改善，但没有 光感受器的缓慢退化。这项以基因治疗为导向的提案的目标是确定 治疗是可以在已经患病的视网膜的背景下实现的，以及基因治疗应该在什么时候进行 用于改善视杆细胞和视锥细胞营养不良患者的功能视力。 在之前的资助期间，我们成功地恢复了视杆细胞模型的小鼠视网膜功能 退化，表明只需要12%的弹性光感受器来拯救。我们现在打算 确定这一阈值是否可以转移到更低数量的弹性光感受器(6%)，以实现功能性 幻象。我们还将确定在疾病的晚期是否有可能进行锥体特异性抢救。最后，我们会 确定对杆状单色症进行基因治疗的最新时间点。为了做到这一点，我们 将在锥体特异性G蛋白中使用一种新的、可诱导的遗传救援系统，即结合α的鸟嘌呤核苷酸。 转导蛋白2(Gnat2)，这将允许我们有条件地逆转GNA2缺乏，同时控制颞叶 以及表型反转的空间方面。我们还将使用磷酸二酯酶6(PDE6)小鼠作为 杆状营养不良。 Gnat2flxSTOP：：Arr3CreERT2/WT和Pde6flxSTOP/PdeloxSTOP：：Arr3CreERT2/WT可编程型号将 提供一个平台，为旨在增加以下视觉功能恢复的持续努力做出贡献 杆状和视锥细胞介导的营养不良的基因治疗。它们还将使我们能够解决几个引人注目的、 临床相关问题：大脑回路是否足够可塑性，可以从病理变化中恢复 是由Gnat2突变引起的？有没有一个不归路的点，在那之后，尽管基因恢复到 野生型，球果不能打捞吗？新陈代谢能否消除基因治疗的时间障碍 重新编程？ 综上所述，这项建议肯定会1)界定限制介入治疗的因素；2)验证一种新的、 视杆细胞和视锥细胞介导的视网膜变性的可诱导模型；以及3)决定代谢是否 重编程可以作为治疗视网膜变性的一种有效的、非基因特异性的策略。

PROJECT SUMMARY Of the retinal degenerative diseases that affect 9 million Americans, rod and cone photoreceptor dystrophies are arguably the most devastating. Gene therapy is a potential means to strengthen photoreceptor viability. However, the first human gene therapy trial for retinal degeneration 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 and at what timepoints gene therapy should be administered in order to improve functional vision in rod and cone dystrophies. During the previous funding period, we succeeded in restoring retinal function in mouse model of rod degeneration, demonstrating that only 12% of resilient photoreceptors are needed for rescue. We now intend to determine if this threshold may be shifted to an even lower number of resilient photoreceptors (6%) for functional vision. We will also determine whether cone-specific rescue is possible at late stages of disease. Finally, we will determine the latest timepoint at which gene therapy can be administered for rod monochromatism. To do this, we will use a novel, inducible genetic rescue system in the cone-specific G-protein, guanine nucleotide binding α- transducin 2 (Gnat2), which will allow us to conditionally reverse GNAT2-deficiency while controlling the temporal and spatial aspects of phenotypic reversal. We will also use a phosphodiesterase 6 (Pde6) mouse as a model for rod dystrophy. The Gnat2floxSTOP/ Gnat2floxSTOP::Arr3CreERT2/WT and Pde6floxSTOP/PdefloxSTOP::Arr3CreERT2/WT programmable models will provide a platform for contributing to ongoing efforts aimed at increasing restoration of visual function following gene therapy for rod- and cone-mediated dystrophies. They will also allow us to address several compelling, clinically relevant questions: Is the brain’s circuitry sufficiently plastic to recover from the pathological changes caused by the Gnat2 mutation? Is there a point of no return after which, despite reversion of the genotype to wild type, cones cannot be salvaged? Can temporal barriers to gene therapy be relieved by metabolic reprogramming? Taken together, this proposal is certain to 1) define the factors limiting interventional therapy; 2) validate a new, inducible models of rod- and cone-mediated retinal degeneration; and 3) determine whether metabolic reprogramming can serve as an efficacious, non-gene-specific strategy for treating retinal degeneration.