Identification of genes related to spcific properties of mammalian cells

背景：哺乳动物细胞目前是人类治疗和诊断所需的各种生化化合物的主要产生者。这些细胞具有独特的特性，如生长速度慢和依赖锚定的行为，这影响了生产效率。我们的研究工作集中在识别影响生产性能的基因和microRNA。通过利用这些基因和microRNA，有可能改变细胞的行为，提高它们的生产能力。 在过去的几年里，我们证明了通过使用非编码RNA，特别是microRNAs和siRNAs来操纵基因表达来改善哺乳动物细胞的生长和重组蛋白的生产是可能的。我们开始使用这种方法处理特定的microRNA，并继续执行高通量技术，分析包含数百个microRNA和数千个siRNA的文库。通过对22000个基因的高通量siRNA分析，确定了10个抑制促进重组蛋白表达的基因。其中OAZ1-编码鸟氨酸脱羧酶抗酶1的基因-被选中进行详细研究，因为它的沉默提高了报告蛋白的产量，而不影响细胞活力。沉默OAZ1导致鸟氨酸脱羧酶表达增加，细胞内腐胺和亚精胺水平增加。研究表明，OAZ1是提高重组蛋白表达水平的新靶点。这项工作中进行的基因组规模的筛选可以为为各种生物技术应用而有针对性地设计高效的哺乳动物细胞平台奠定基础。本项目的下一步是建立OAZ1基因缺失的HEK细胞系。利用Crispr技术建立了缺失OAZ1的HEK细胞系，在不影响生长和代谢活性的情况下，荧光素酶的表达增加了三倍。这项工作继续进行，删除了包括在10个基因列表中的CASP8AP2基因。我们通过CRISPR/Cas9基因组编辑敲除CASP8AP2基因，并评估其敲除对重组蛋白表达、细胞生长、细胞活力和整体基因表达的影响，从而验证CASP8AP2基因在HEK293细胞中作为工程靶点的有效性。在不含CASP8AP2的HEK293细胞中，重组荧光素酶的特异性表达增加了7倍，重组SEAP的特异性表达增加了2.5倍，而对细胞的生长和活力没有明显的影响。转录组分析表明，细胞周期的下调，特别是细胞周期蛋白依赖的激酶抑制因子2A(CDKN2A)基因的上调，有助于CASP8AP2基因缺陷细胞中重组蛋白表达的改善。这些结果验证了CASP8AP2基因是提高重组蛋白在HEK293细胞中表达的可行的工程靶点。 除了蛋白质表达外，哺乳动物细胞也是病毒表达的来源。因此，根据我们利用非编码RNA提高蛋白质生产的经验，我们希望确定miRNA或siRNA物种与病毒结构蛋白和辅助蛋白共同表达，以影响rAAV滴度的增加，而不依赖于所传递的转基因。我们预计病毒生产将受益于不同的优化miRNA图谱。到目前为止，我们的工作一直是筛选条件的精炼。筛选可以被认为是单独的转导/生产和转导/检测步骤。选择不同的转染参数(平板密度、质粒浓度、转染剂和正反向转染法的天数)来优化(I)在293T/17细胞中ITRs的荧光蛋白表达，以及(Ii)在COS7细胞中的相应转导也通过荧光蛋白表达来评估。作为补充，选择转导参数以允许(I)在添加相同的上清液批次的情况下获得最高的荧光信号，(Ii)最小化液体处理误差，以及(Iii)对于显著低于非饱和的MOI/信号水平。相关参数的例子包括HEK293T/17细胞板密度(3e3)、质粒总数(0.12g)、反向和正向转染剂(Dharmafet4和Viafect)以及用于评估转导的细胞系(COS7)。根据初步结果，改进信号和最小化变异性的努力包括增加反向和正向转染之间的时间，增加更多的自动化步骤，转向荧光素酶作为检测标记，以及在筛选中添加新的特异性siRNA文库。这一优化过程继续进行，并确定了更多影响高通量分析的参数。这项工作是与NCAT共同完成的。

Background: Mammalian cells are currently the main producers of various biochemical compounds needed for human therapeutic and for diagnostic purposes. These cells have distinct properties, such as slow growth rate and anchorage-dependent behavior, which affect production efficiency. Our research work is concentrating on identification of genes and microRNA that affects production properties. By utilizing these genes and microRNAs it be likely possible to change the cells behavior and improve their production capability. In the last several years we demonstrated that it is possible to improve mammalian cells growth and recombinant protein production by manipulating gene expression using noncoding RNAs, especially microRNAs and siRNAs. We initiated this approach working with specific microRNAs and we continued performing high throughput techniques, analyzing libraries containing hundreds of microRNAs and thousands of siRNAs. By conducing high throughput siRNA analysis of 22000 genes, 10 genes whose inhibition improved recombinant protein expression were identified. Among them OAZ1-the gene encoding the ornithine decarboxylase antizyme1- which was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of ornithine decarboxylase expression with increased cellular levels of putrescine and spermidine. The study demonstrated that OAZ1 is a novel target for improved expression of recombinant proteins. The genome-scale screening performed in this work can establish a foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Creating HEK cell line in which the OAZ1 gene was deleted was the next step in this project. By using Crispr technology HEK cell line lacking OAZ1 was created, that showed three times increased expression of luciferase without affecting growth and metabolic activities. The work continued by deleting the CASP8AP2 gene that was included in the 10 genes list. We validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed 7-fold increase in specific expression of recombinant luciferase and a 2.5-fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that de-regulation of the cell cycle, specifically the upregulation of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line. In addition to protein expression Mammalian cells are the source for virus expression. So, based on our experience utilizing noncoding RNA for improved protein production we looked to identify miRNA or siRNA species to co-express alongside viral structural and helper proteins to affect an increase in rAAV titer, independent of the transgene being delivered. We expect viral production to benefit from a different optimized miRNA profile. Our work thus far has been the refining of screening conditions. The screen can be considered as separate transfection/production and a transduction/assay step. Transfection parameters (plating density, plasmid concentration, transfection reagent, and days between reverse and forward transfection) were chosen to optimize (i) fluorescent protein expression from the ITRs in 293T/17 cells as well as (ii) corresponding transduction seen in COS7 cells also assessed via fluorescent protein expression. Complementarily, transduction parameters were chosen to allow for (i) the highest fluorescent signal given the addition of the same supernatant batches, (ii) minimizing liquid handling error, and (iii) for a level of MOI/signal significantly less than non- saturating. Examples of relevant parameters include HEK293T/17 cell plating density (3e3), total plasmid amount (0.12 g), the reverse and forward transfection reagents (Dharmafect4 and Viafect), and the cell line to evaluate transduction (COS7). Based on initial results, efforts to improve the signal and minimize variability have included increasing the time between reverse and forward transfection, adding more automation steps, moving to luciferase as the assay marker, and adding new specific siRNA libraries to the screen. This optimization process was continued and more parameters affecting the high throughput analysis were identified. this work was done together with NCAT.