Defining Barriers to Gene Therapy

项目摘要 在影响9百万美国人的视网膜变性疾病中， 可以说是最具破坏性的。基因治疗是增强感光细胞活力的潜在手段。 然而，第一个针对视网膜变性的人类基因治疗试验发现， 感光器的缓慢退化。这个基因治疗导向的提案的目标是确定是否 在已经患病的视网膜的情况下，基因治疗是可以实现的，并且在什么时间点基因治疗应该是可行的。 施用以改善视杆细胞和视锥细胞营养不良中的功能性视力。 在上一个资助期间，我们成功地恢复了视网膜功能的小鼠模型杆 退化，表明只有12%的弹性光感受器需要救援。我们现在打算 确定这个阈值是否可以转移到甚至更低数量的弹性光感受器（6%）， 视野我们还将确定在疾病的晚期是否可能进行视锥细胞特异性拯救。最后我们将 确定基因治疗可以用于视杆细胞单色性的最后时间点。为此我们 将使用一种新的，可诱导的遗传拯救系统，在锥特异性G蛋白，鸟嘌呤核苷酸结合α- 转导蛋白2（Gnat 2），这将使我们能够有条件地逆转GNAT 2缺陷，同时控制时间 以及表型逆转的空间方面。我们还将使用磷酸二酯酶6（Pde 6）小鼠作为模型， 杆营养不良 Gnat 2floxSTOP/Gnat 2floxSTOP：：Arr 3CreERT 2/WT和Pde 6 floxSTOP/PdefloxSTOP：：Arr 3CreERT 2/WT可编程模型将 提供一个平台，为正在进行的旨在促进视觉功能恢复的努力做出贡献， 视杆细胞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令人信服的， 临床相关问题：大脑回路是否具有足够的可塑性，能够从病理变化中恢复 是由Gnat 2突变引起的吗有没有一个不归点，在此之后，尽管基因型逆转， 野生型，锥不能挽救？基因治疗的时间障碍是否可以通过代谢治疗来缓解 重新编程 总之，这一建议肯定会1）定义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;和3）确定是否代谢 重编程可以作为治疗视网膜变性的有效的、非基因特异性策略。

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.