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

1. NTBC在OCA 2、OCA 3和OCA 4小鼠模型中的作用 我们先前发表的工作已经确定尼替西酮（NTBC）可以增加OCA 1B小鼠模型中的黑色素沉着（Onojafe等人，投资眼科维斯科学2018年）。然而，用口服NTBC治疗OCA 3小鼠模型对黑色素产生没有有利的影响，并且对虹膜基质中色素沉着黑素体的数量影响最小（Onojafe et al.，投资眼科维斯科学2019年）。 我们进一步假设NTBC可能改善OCA 2（黑素细胞特异性转运蛋白）和OCA 4（SLC 45 A2的所谓“白下”等位基因）小鼠模型的黑化。在Nitisinone治疗一个月后，NTBC治疗的OCA 2和OCA 4小鼠的血浆酪氨酸浓度增加，没有明显的毒副作用。在两个小鼠系中，被毛色素沉着均增加。虹膜色素沉着主要在OCA 4小鼠中增加。电子显微镜检查也证实脉络膜色素沉着略有增加，而在两条线的RPE中均未观察到影响（手稿正在编写中）。 2.高通量药物筛选以鉴定调节Tyr活性的化合物 我们在高通量药物筛选中使用纯化的截短的Tyr蛋白（先前测试并验证具有与全长蛋白等同的酶活性）。与NCATS合作，我们从Genesis Drug Collection，Natural Products Library和NCATS Pharmaceutical Collection中筛选了34，000种化合物。我们鉴定了超过100种新的酪氨酸酶抑制剂和一些激活剂。在体外二次酶筛选和斑马鱼体内验证后，我们在OCA 1B小鼠模型上测试了三种剂量的顶级候选化合物。当腹膜内给药30天时，药物耐受性良好。初步分析表明，接受治疗的小鼠的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的体外培养皿中疾病模型 我们利用诱导多能干细胞（iPSC）技术建立了眼皮肤白化病1A型（OCA 1A）和2型（OCA 2）的培养皿中疾病模型。将OCA患者成纤维细胞重编程为iPSC，并使用发育指导的分化方案分化为视网膜色素上皮（OCA-RPE）。组织形态学和生理学以及视觉周期装置的表达和功能与对照着色的iPSC衍生的RPE相当。此外，在OCA-RPE细胞中包含不成熟黑素体的色素沉着缺陷在体外忠实地复制了体内组织特征。我们目前正在测试基因替换和CRISPR介导的基因编辑方法，目的是恢复OCA-RPE的表型和色素沉着。 4. 脉络膜上基因替代疗法 我们目前正在进行的初步研究脉络膜上交付的AAV-酪氨酸酶基因治疗构建体的大鼠模型眼皮肤白化病，1型。

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.