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

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.