Transcriptomic RNAseq drug screen in cerebrocortical cultures; towards novel neurogenetic disease therapies.

Rare monogenic diseases affect millions worldwide; although over 4,500 rare disease genotypes are known, disease-modifying drugs are available for only 5% of them. The sheer number of these conditions combined with their rarity precludes traditional costly drug discovery programs. An economically viable alternative is to repurpose established drugs for rare diseases. Many genetic diseases result from increased or decreased protein activity and identification of clinically approved drugs which moderate this pathogenic dosage holds therapeutic potential. To identify such agents for neurogenetic diseases, we have generated genome-wide transcriptome profiles of mouse primary cerebrocortical cultures grown in the presence of 218 blood brain barrier penetrant clinic-tested drugs. RNAseq and differential expression analyses were used to generate transcriptomic profiles; therapeutically relevant drug-gene interactions related to rare neurogenetic diseases identified in this fashion were further analyzed by qRT-PCR, western blot and immunofluorescence. We have created a transcriptome-wide searchable database for easy access to the gene expression data resulting from the cerebrocortical drug screen (Neuron Screen) and have mined this data to identify a novel link between thyroid hormone and expression of the peripheral neuropathy associated gene Pmp22. Our results demonstrate the utility of cerebrocortical cultures for transcriptomic drug screening, and the database we have created will foster further discovery of novel links between over 200 clinic-tested blood brain barrier penetrant drugs and genes related to diverse neurologic conditions. Overall design: mRNA differential expression profiles of mouse primary cortical cultures treated individually with 218 clinic-tested drugs and 9 vehicle treated samples, 1 replicate performed per drug.

罕见单基因遗传病（rare monogenic diseases）影响全球数百万人群；尽管目前已鉴定出超过4500种罕见病基因型，但仅有5%的病种拥有疾病修饰药物（disease-modifying drugs）。这类疾病数量庞大且发病率极低，使得传统高成本药物研发项目难以实施。经济可行的替代方案是对已上市药物进行罕见病适应症再利用。许多遗传病源于蛋白质活性异常升高或降低，筛选出可调节该致病性蛋白剂量的临床获批药物，将具备治疗潜力。为识别针对神经遗传病的此类药物，我们构建了在218种可穿透血脑屏障（blood brain barrier）且经临床测试的药物干预下培养的小鼠原代大脑皮层细胞的全基因组转录组谱。通过RNA测序（RNAseq）与差异表达分析生成转录组谱；以此方式筛选得到的与罕见神经遗传病相关的治疗性药物-基因相互作用，进一步通过实时荧光定量PCR（qRT-PCR）、蛋白质免疫印迹（western blot）与免疫荧光（immunofluorescence）进行验证分析。我们构建了一个可检索的全转录组数据库，方便获取该大脑皮层药物筛选实验（Neuron Screen）产生的基因表达数据，并通过挖掘该数据发现了甲状腺激素与周围神经病相关基因Pmp22表达之间的新型关联。本研究结果证实了大脑皮层原代细胞培养用于转录组药物筛选的实用性，而我们构建的数据库将助力进一步发掘200余种经临床测试的血脑屏障穿透性药物与多种神经系统疾病相关基因之间的新型关联。总体实验设计：对分别用218种经临床测试药物及9份溶剂对照处理的小鼠原代皮层细胞进行mRNA差异表达谱分析，每种药物设置1个生物学重复。