Pre-clinical and clinical studies of NTBC as a potential treatment for albinism

我们之前报道的关于OCA1A和OCA1B小鼠模型的工作已经发表在《临床调查杂志》上。这项工作证实了烟替松可以增加OCA1B小鼠模型中的黑色素沉着，但不能增加OCA1A的黑色素沉着。 自上次报告以来，我们侧重于以下分项目： 1.经NTBC处理的OCA1B小鼠与对照组比较的视觉功能 我们已经通过几种方法评估了尼替松治疗对视网膜和视觉功能的影响。与钱博士合作，我们已经确定了尼替松治疗对全场ERG(暗视和明视)的影响。在与Anand Swaroop博士团队成员的合作下，我们正在评估产前/出生后早期尼替松治疗对视网膜神经发育的影响。与Tudor Badea博士的团队合作，我们研究了尼替松治疗对空间视觉敏感度和神经回路发育的影响。我们目前正在准备一份手稿以供出版。 2烟替松对OCA3小鼠模型的影响 我们推测尼替松可能会改善OCA3小鼠模型(Tyrp1棕色小鼠)的黑化，因为已知酪氨酸酶和TYRP-1在黑素体膜上相互作用并稳定。从杰克逊实验室收到的小鼠似乎携带了一种新的无意义的Tyrp1等位基因，而不是典型的棕色等位基因。我们在临床上研究了烟替松对小鼠眼部色素沉着和毛发的影响，并研究了其对OCA3小鼠眼部色素组织中黑素小体数量/面积的影响。这些数据正在准备公布。 3.烟替松对OCA4小鼠模型的影响 我们假设尼替米诺可能会改善OCA4(SLC45A2的“白下”等位基因)小鼠模型中的黑化。来自杰克逊实验室的小鼠，尽管它们都有典型的白下等位基因，但当培育成纯合子时，它们表现出两种截然不同的毛色(“浅”和“深”)。我们目前正在评估这两种OCA4小鼠的黑化细胞的特征，并正在使用高通量测序来识别第二个，修改的等位基因。同时，我们还在进行治疗实验，以确定尼替松是否会改善该模型的两个亚型的眼睛/毛皮色素沉着。 4.外用尼替松和沙拉坦治疗眼部色素沉着 我们目前正在确定尼替松作为局部滴眼剂是否能够改善喜马拉雅(OCA1B)小鼠眼睛中的黑色素含量。与此同时，我们还在测试FDA批准的药物沙拉坦，该药物已被证明可以增加服用这种局部滴眼剂治疗青光眼的患者的虹膜色素沉着。 5.高通量药物筛选，寻找调节酪氨酸酶活性的化合物 我们在幼虫表达系统中纯化了重组野生型和突变型人酪氨酸酶，并进行了详细的酶学研究。这些数据已发表在《公共科学图书馆·综合》上。我们建立了一种测定酪氨酸酶活性的荧光测定法。与NCATS的Marc Ferrer博士和他的团队合作，我们正在启动一项药物筛选，以确定在体外可能抑制或增强酪氨酸酶活性的化合物。我们已经开发了一条验证靶点的管道，包括酶功能和靶向的体外研究。在这些屏幕之后将是基于动物的屏幕(即斑马鱼，最终是喜马拉雅老鼠)。 6.尼替西酮治疗OCA1B患者的临床方案 我们已经建立了IRB批准的方案，用于测试标准剂量的尼替松对OCA1B患者眼部和全身黑色素沉着的影响。自上一份年度报告以来，招聘工作出人意料地困难。尽管我们对几个人进行了筛查，但他们不符合纳入//排除标准。我们继续对全国各地的患者进行筛查。

Our previously-reported work on OCA1A and OCA1B mouse models has been published in the Journal of Clinical Investigation. This work established the nitisinone could increase melanin pigmentation in a mouse model of OCA1B, but not OCA1A. Since the time of the last report, we have focused on the following sub-project: 1. Visual function in NTBC-treated OCA1B mice vs. controls We have evaluated the effect of nitisinone treatment on the retinal and visual function using several approaches. In collaboration with Dr. Qian, we have characterized the effect of nitisinone treatment on the full-field ERG (scotopic and photopic). In collaboration with members of Dr. Anand Swaroop's group we are assessing the effect of prenatal/early postnatal nitisone treatment on neurla retina development. In collaboration with Dr. Tudor Badea's group, we have studied the effect of nitisinone treatment on spatial visual acuity and on neural circuit development. We are currently preparing a manuscript for publication. 2 Effect of nitisinone on a mouse model of OCA3 We hypothesized that nitisinone might improve melanization in a mouse model of OCA3 (the Tyrp1-brown mouse), as tyrosinase and TYRP-1 are known to interact and stabilize one another in the melanosome membrane. The mouse received from Jackson Labs appears to harbor a novel nonsense allele of Tyrp1, rather than the canonical brown allele. We have studied the effect of nitisinone treatment on fur and eye pigmentation clinically and have studed its effect on melanosome number/area in the pigmented tissues of the OCA3 mouse eye. These data are being prepared for publication. 3. Effect of nitisinone on a mouse model of OCA4 We hypothesized that nitisinoen might improve melanization in a mouse model of OCA4(the so-called "underwhite" allele of SLC45A2). The mice received from Jackson Lab, although they all harbor the classic underwhite allele, demonstrateed two distinct coat colors ("light" and "dark")when bred to homozygosity. We are currently evaluating the characteristics of melanized cells in both varieties of OCA4 mouse and are using high-throughput sequencing to identify the second, modifying allele. Simultaneously, we are also conducting treatment experiments to determine if nitisinone will improve ocular/fur pigmentation in both sub-strains of this model 4. Use of topical nitisinone and Xalatan in ocular pigmentation We are currently determining whether nitisinone, when delivered as a topical eye drop, is capable of improving melanin content in the eyes of Himalayan (OCA1B) mice. In parallel, we are also testing the FDA-approved drug, Xalatan, which has been shown to increase iris pigmentation in patients taking this topical drop for glaucoma. 5. High-throughput drug screening to find compounds that regulate tyrosinase activity We have purified recombinant wild-type and mutant human tyrosinase in a larval expression system and performed detailed enzymology. These data have been published in PLoS ONE. We have established a fluorometric assay for measuring tyrosinase activity. In collaboration with Dr. Marc Ferrer and his team at NCATS, we are initiating a drug screen to identify compounds that may inhibit or enhance tyrosinase activity in vitro. We have developed a pipeline for validating targets, including in vitro studies of enzyme function and targeting. These screens will be followed by animal-baesd (i.e., zebrafish and, eventually, Himalayan mouse) screens. 6. Clinical Protocol for Studying the Effect of Nitisinone Treatment in Human Subjects with OCA1B We have established an IRB-approved protocol for the testing of a standard oral dose of nitisinone on ocular and systemic melanin pigmentation in patients with OCA1B. Recruitment has bee unexpectedly difficult since the time of the last annual report. Although we have screened several individuals, they did not meet inclusion//exclusion criteria. We continue to screen patients from around the country.