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

我们之前报道的OCA 1A和OCA 1B小鼠模型的工作已发表在《临床研究杂志》上。 这项工作建立了nitisinone可以增加OCA 1B小鼠模型中的黑色素沉着，但不是OCA 1A。 自上一次报告以来，我们重点关注以下次级项目： 1. NTBC处理的OCA 1B小鼠与对照小鼠的视觉功能 我们已经使用几种方法评估了尼替西酮治疗对视网膜和视功能的影响。 在与钱博士的合作中，我们描述了尼替西酮治疗对全视野ERG（暗视和明视）的影响。 与Anand Swaroop博士的小组成员合作，我们正在评估产前/产后早期尼替松治疗对神经视网膜发育的影响。 与Tudor Badea博士的小组合作，我们研究了尼替西酮治疗对空间视觉敏锐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尼替西酮对OCA 3小鼠模型的作用 我们假设，nitisinone可能会改善小鼠模型中的OCA 3（酪氨酸蛋白1-棕色小鼠）的黑化，因为酪氨酸酶和TYRP-1是已知的相互作用和稳定在黑素体膜。 从杰克逊实验室接收的小鼠似乎具有Tyrp 1的新型无义等位基因，而不是典型的棕色等位基因。 我们已经研究了尼替西酮治疗对临床毛皮和眼睛色素沉着的影响，并研究了其对OCA 3小鼠眼睛色素组织中黑素小体数量/面积的影响。 这些数据正在准备出版。 3. 尼替西酮对OCA 4小鼠模型的影响 我们假设nitisinoen可能会改善OCA 4小鼠模型（SLC 45 A2的所谓“underwhite”等位基因）的黑化。 从杰克逊实验室接收的小鼠，尽管它们都携带经典的白下等位基因，但当繁殖到纯合性时，表现出两种不同的毛色（“浅色”和“深色”）。 我们目前正在评估两种OCA 4小鼠中黑化细胞的特征，并使用高通量测序来鉴定第二个修饰等位基因。 同时，我们也在进行治疗实验，以确定尼替西酮是否会改善该模型的两个亚株的眼/皮毛色素沉着 4. 尼替西酮和Xalatan在眼部色素沉着中的应用 我们目前正在确定尼替西酮作为局部滴眼液是否能够改善喜马拉雅（OCA 1B）小鼠眼睛中的黑色素含量。 与此同时，我们也在测试FDA批准的药物Xalatan，该药物已被证明会增加青光眼患者的虹膜色素沉着。 5. 高通量药物筛选以发现调节酪氨酸酶活性的化合物 我们纯化了重组野生型和突变体人酪氨酸酶的幼虫表达系统，并进行了详细的酶学。 这些数据发表在PLoS ONE上。 我们建立了一种荧光法测定酪氨酸酶活性。 与NCATS合作，我们正在启动一项药物筛选，以确定可能抑制或增强酪氨酸酶体外活性的化合物。 我们已经开发了一个验证目标的管道，包括酶功能和靶向的体外研究。 这些屏幕之后将进行基于动物的（即，斑马鱼和喜马拉雅小鼠）筛选。 6. 研究尼替西农治疗OCA 1B人类受试者效果的临床方案 我们已经建立了IRB批准的方案，用于测试标准口服剂量的尼替西酮对OCA 1B患者眼部和全身黑色素沉着的影响。 自去年的报告以来，我们已经完全招募了所有5名OCA 1B患者并开始治疗。 未发生严重不良事件。 我们将继续通过预先确定的结果变量监测治疗。

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 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-based (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. Since last year's report, we have fully enrolled all five patients with OCA1B and begun treatment. No serious adverse events have occurred. We are continuing to monitor treatment via predetermined outcome variables.