Yau Edwin H, Butler Mark C, Sullivan Jack M
Department of Pharmacology/Toxicology, University at Buffalo- SUNY, Buffalo, NY 14209, USA; Department of Ophthalmology (Ira G. Ross Eye Institute), University at Buffalo- SUNY, Buffalo, NY 14209, USA.
Department of Ophthalmology (Ira G. Ross Eye Institute), University at Buffalo- SUNY, Buffalo, NY 14209, USA.
Exp Eye Res. 2016 Oct;151:236-55. doi: 10.1016/j.exer.2016.05.020. Epub 2016 May 25.
Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.
治疗性转录后基因沉默（PTGS）药物（如核酶、RNA干扰、反义核酸）研发中的主要瓶颈包括：确定mRNA靶标的罕见可及区域（可供退火和切割）面临挑战；在人类细胞中对药物进行测试和优化以确定先导药物；检测细胞毒性；以及在合适的疾病动物模型中进行临床前评估。需要快速且可靠的细胞检测方法来确定用于优化的有效先导候选药物。我们的目标是开发一种快速评估多种RNA药物的方法，以确定与疾病状态相关的特定mRNA的先导候选药物。我们开发了一种基于人类细胞的快速筛选平台，使用视网膜变性模型靶标——人类视杆细胞视蛋白（RHO）mRNA，来测试锤头状核酶（hhRz）或RNA干扰（RNAi）构建体的功效。重点是针对多种视网膜变性靶标的RNA药物发现。为了验证该方法，根据mRNA可及性的计算机预测结果，针对分泌性碱性磷酸酶（SEAP，一种模型基因表达报告基因）的靶标mRNA中的NUH↓切割位点（N = G、C、A、U；H = C、A、U）测试候选hhRz。将hhRz嵌入更大的稳定腺病毒VAI RNA支架中，以实现高细胞表达、细胞质运输和稳定性。当通过用于高通量筛选（HTS）的荧光酶测定法轻松检测时，大多数hhRz表达质粒对细胞外SEAP酶活性具有统计学上显著的敲低作用。利用SEAP报告基因可在活细胞中测量细胞靶标的PTGS敲低动力学。经过验证的SEAP HTS平台被用于确定针对遗传性视网膜变性模型靶标RHO mRNA的先导PTGS药物。采用了两种方法将视网膜基因模型靶标mRNA与SEAP报告基因mRNA进行物理融合。评估大量针对不同NUH↓切割位点的潜在VAI-hhRz表达质粒的最便捷方法是使用稳定表达双顺反子Target-IRES-SEAP靶标融合mRNA的培养人类HEK293S细胞。这种合理的RNA药物发现方法对于任何眼科疾病相关的mRNA靶标以及一般的任何疾病mRNA靶标都具有广泛的实用性。该方法将允许根据效力对PTGS药物进行排序，以确定用于进一步优化的先导治疗化合物。
