Pre-clinical and clinical studies of NTBC and other compounds as potential treatments for albinism

1.NTBC对OCA2、OCA3、OCA4小鼠模型的影响 我们之前发表的工作已经证实，烟替松(NTBC)可以增加OCA1B小鼠模型中的黑色素沉着(Onojafe等人，Invest Ophthalol Vis Sci)。2018年)。然而，口服NTBC治疗OCA3小鼠模型并没有对黑色素的产生产生有利的效果，并且对虹膜基质中有色黑素小体的数量影响最小(Onojafe等人，Invest Ophthalol Vis Sci)。2019年)。 我们进一步假设，NTBC可能会改善OCA2(黑素细胞特异性转运蛋白)和OCA4(SLC45A2的“白下”等位基因)小鼠模型的黑化。NTBC处理的OCA2和OCA4小鼠在尼替松治疗一个月后血浆酪氨酸浓度升高，没有明显的毒副作用。两个品系的小鼠皮毛色素沉着都增强了。虹膜色素沉着主要在OCA4小鼠身上增强。电子显微镜也证实了脉络膜色素沉着的轻微增加，而在两个品系的RPE中没有观察到影响(手稿正在准备中)。 2.高通量药物筛选以确定调节Tyr活性的化合物 我们在高通量药物筛选中使用了纯化的、截短的Tyr蛋白(之前经过测试和验证具有与全长蛋白相同的酶活性)。与NCATS合作，我们从Genesis药物收藏、天然产品图书馆和NCATS药物收藏中筛选出34,000种化合物。我们确定了&gt；100种新的酪氨酸酶抑制剂和一些激活剂。在体外和体内的二次酶筛中验证后，我们在OCA1B小鼠模型上测试了三种剂量的首选候选化合物。这种药物在ip时耐受性良好。30天。初步分析表明，接受治疗的小鼠头发黑色素略有增加，但眼睛黑色素没有令人信服的变化。 3.OCA体外病盘模型的建立 我们利用诱导多能干细胞(IPSC)技术建立了1A型眼皮肤白化病(OCA1A)和2型眼皮肤白化病(OCA2)的盘中病模型。OCA患者的成纤维细胞被重新编程为IPSCs，并使用发育指导的分化方案向视网膜色素上皮(OCA-RPE)分化。组织形态和生理学以及视觉周期装置的表达和功能与对照着色IPSC来源的RPE相似。此外，体外培养的OCA-RPE细胞中含有未成熟黑素小体的色素沉着缺陷忠实地复制了体内的组织特征。我们目前正在测试基因替换和CRISPR介导的基因编辑方法，目标是逆转OCA-RPE的表型和恢复色素沉着。 4.脉络膜上基因替代疗法 我们目前正在进行脉络膜上脉络膜上递送AAV-酪氨酸酶基因治疗构建体在1型眼皮肤白化大鼠模型中的初步研究。

1. Effect of NTBC in mouse models of OCA 2, OCA3 and OCA4 Our previously-published work had established that nitisinone (NTBC) can increase melanin pigmentation in a mouse model of OCA1B (Onojafe et al., Invest Ophthalmol Vis Sci. 2018). However, treatment of a mouse model of OCA3 with oral NTBC does not have a favorable effect on melanin production and minimally affects the number of pigmented melanosomes in the iris stroma (Onojafe et al., Invest Ophthalmol Vis Sci. 2019). We further hypothesized that NTBC might improve melanization in mouse models of OCA2 (melanocyte-specific transporter protein) and of OCA4 (the so-called "underwhite" allele of SLC45A2). Plasma tyrosine concentration was increased in NTBC-treated OCA2 and OCA4 mice after a month of nitisinone treatment with no overt toxic side-effects. Fur pigmentation was augmented in both mouse lines. Iris pigmentation was augmented predominantly in OCA4 mice. Electron microscopy also confirmed a small increase in choroidal pigmentation, whereas, no effect was observed in the RPE in both lines (manuscript in preparation). 2. High-throughput drug screening to identify compounds that regulate Tyr activity We used purified, truncated Tyr protein (previously tested and validated to have equivalent enzymatic activity to full length protein) in a high-throughput drug screening. In collaboration with NCATS, we screened 34,000 compounds from the Genesis Drug Collection, the Natural Products Library, and the NCATS Pharmaceutical Collection. We identified >100 new inhibitors and a few activators of tyrosinase. After validation in a secondary enzymatic screen in vitro and in zebrafish in vivo, we have tested three doses of the top candidate compound on the OCA1B mouse model. The drug was well tolerated when administered i.p. for 30 days. Preliminary analysis indicated a modest increase in hair melanin in treated mice, but no convincing changes in eye melanin. 3. In vitro disease-in-a-dish modeling of OCA We developed a disease-in-a-dish model of oculocutaneous albinism type 1A (OCA1A) and type 2 (OCA2) using induced pluripotent stem cell (iPSC) technology. OCA patient fibroblasts were reprogrammed to iPSCs and differentiated to retinal pigment epithelium (OCA-RPE) using a developmentally-guided differentiation protocol. Tissue morphology and physiology as well as expression and functionality of the visual cycle apparatus were comparable to control pigmented iPSC-derived RPE. Furthermore, pigmentation defects comprising immature melanosomes in the OCA-RPE cells in vitro faithfully replicated tissue characteristics in vivo. We are currently testing gene replacement and CRISPR-mediated gene editing approaches with the goal to revert the phenotype and restore pigmentation in OCA-RPE. 4. Suprachoroidal gene replacement therapy We are currently conducting pilot studies of suprachoroidal delivery of an AAV-tyrosinase gene therapy construct in a rat model of oculocutaneous albinism, type 1.