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

1.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OCA1B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。 我们与康奈尔大学的Jonathan Zippin博士合作扩展了这些研究，他使用HPLC来测量药物治疗后黑色素合成的通量。 我们目前正在使用体外模型来验证我们是否看到皮肤黑色素对药物的反应发生了变化。 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（OCA1A）和2型（OCA2）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介导的基因编辑以及小分子方法，目的是恢复OCA-RPE的表型和色素沉着。进一步的研究着眼于自噬在黑素体生物学中的作用，与我们在白化病细胞中观察到的异常相比。 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. 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. We have extended these studies in collaboration with Dr. Jonathan Zippin at Cornell, who has used HPLC to measure flux in melanin synthesis as a result of drug treatment. We are currently using an in vitro model to validate whether we see a change in skin melanin in response to drug. 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 as well as small molecule approaches with the goal to revert the phenotype and restore pigmentation in OCA-RPE. Further studies have looked at the role of autophagy in melanosome biology vis-a-vis the abnormalities we observe in albinism cells. 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.