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，如果代谢重新编程可以是一种 有效的治疗选择。 在之前的资助期间，我们成功地恢复了11个月以上的视网膜功能 在小鼠的杆状变性模型中，即使在变性开始和疾病晚期也是如此。我们现在 旨在确定同样的结果是否可以在视锥细胞营养不良症中实现。要做到这一点，我们将 在锥体特异性G蛋白中产生一个新的、可诱导的遗传救援系统，鸟嘌呤核苷酸结合α- 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。 使用Gnat2flxSTOP/Gnat2CreERT2，我们将建立该模型忠实地概括了锥体中介 营养不良(目标1)。我们将在早、中、晚期通过注射他莫昔芬将模型恢复到野生型 疾病分期和评估对退化率的影响(目标2)，以确定 基因疗法。最后，我们将诱导代谢重新编程，并评估其作为一种可能的非基因- 治疗视锥细胞退行性营养不良的具体策略(目标3)。 Gnat2flxSTOP/Gnat2CreERT2可编程模型将提供一个平台，为正在进行的 旨在促进视锥细胞介导的营养不良的基因治疗后视觉功能恢复的努力。 它还将使我们能够解决几个引人注目的、与临床相关的问题：大脑的电路 足够的可塑性来从Gnat2突变引起的病理变化中恢复？是不是有一点 在此之后，尽管基因恢复为野生型，但球果不能被抢救出来？是否可以暂时 基因治疗的障碍可以通过代谢重新编程来消除吗？ 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 视锥细胞介导的视网膜变性的新的、可诱导的模型；以及3)决定代谢重编程 可以作为一种有效的、非基因特异性的治疗视网膜变性的策略。

PROJECT SUMMARY Of the retinal degenerative diseases that affect 9 million Americans, 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 if metabolic reprogramming could be an efficacious treatment option. During the previous funding period, we succeeded in restoring retinal function for more than 11 months in a mouse model of rod degeneration even after the onset of degeneration and at late-stage disease. We now intend to determine whether the same outcomes are achievable in cone-based dystrophies. To do this, we will generate 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. Using Gnat2floxSTOP/Gnat2CreERT2, we will establish that the model faithfully recapitulates cone-mediated dystrophies (Aim 1). We will restore the model to wild type via tamoxifen injection at early, middle, and late disease stages and assess effects on the rate of degeneration (Aim 2) to determine the temporal limitations of gene therapy. Finally, we will induce metabolic reprogramming and assess its utility as a possible non-gene- specific strategy for treating cone degenerations -based dystrophies (Aim 3). The Gnat2floxSTOP/Gnat2CreERT2 programmable model will provide a platform for contributing to ongoing efforts aimed at increasing restoration of visual function following gene therapy for cone-mediated dystrophies. It 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 model of cone-mediated retinal degeneration; and 3) determine whether metabolic reprogramming can serve as an efficacious, non-gene-specific strategy for treating retinal degeneration.