Identification of genes related to spcific properties of mammalian cells

与哺乳动物细胞特定特性相关的基因的鉴定

• 批准号: 10697827
• 负责人: Joseph Shiloach
• 金额: $ 13.7万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10697827
• 关键词: Affect; Automation; Behavior; Biochemical; Biological Assay; Biotechnology; CDKN2A gene; CRISPR/Cas technology; Cell Cycle Regulation; Cell Line; Cell Survival; Cells; Diagnostic; Engineering; Foundations; Gene Expression; Genes; Growth; Human; Investigation; Knock-out; Libraries; Liquid substance; Luciferases; Mammalian Cell; Metabolic; MicroRNAs; National Center for Advancing Translational Sciences; Ornithine Decarboxylase; Plasmids; Production; Property; Proteins; Putrescine; Reagent; Recombinant Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Reporter; Research; Signal Transduction; Small Interfering RNA; Source; Spermidine; Techniques; Therapeutic; Time; Transfection; Transgenes; Untranslated RNA; Up-Regulation; Viral; Virus; Work; base; cell behavior; cell growth; density; design; experience; genome editing; genome-wide; high throughput analysis; improved; novel; process optimization; protein expression; screening; transcriptome

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.

背景资料：哺乳动物细胞目前是人类治疗和诊断目的所需的各种生化化合物的主要生产者。这些细胞具有不同的特性，如缓慢的生长速率和锚定依赖性行为，这影响了生产效率。我们的研究工作集中在鉴定影响生产特性的基因和microRNA。通过利用这些基因和microRNA，有可能改变细胞行为并提高其生产能力。 在过去的几年里，我们证明了通过使用非编码RNA，特别是microRNA和siRNA操纵基因表达来改善哺乳动物细胞生长和重组蛋白生产是可能的。我们开始使用这种方法来研究特定的microRNA，并继续进行高通量技术，分析包含数百种microRNA和数千种siRNA的文库。通过对22000个基因进行高通量siRNA分析，鉴定了10个抑制提高重组蛋白表达的基因。其中OAZ 1-编码鸟氨酸脱羧酶抗酶1的基因-被选择用于详细研究，因为其沉默改善了报告蛋白的产生而不影响细胞活力。沉默OAZ 1引起鸟氨酸脱羧酶表达的增加，同时增加了腐胺和亚精胺的细胞水平。研究表明，OAZ 1是一种新的靶点，用于提高重组蛋白的表达。在这项工作中进行的基因组规模的筛选可以建立一个有效的哺乳动物细胞平台的各种生物技术应用的靶向设计的基础。创建其中OAZ 1基因被删除的HEK细胞系是该项目的下一步。通过使用Crispr技术，创建了缺乏OAZ 1的HEK细胞系，其显示荧光素酶表达增加三倍而不影响生长和代谢活性。这项工作继续删除CASP 8AP 2基因，该基因被列入10个基因列表。我们通过使用CRISPR/Cas9基因组编辑敲除CASP 8AP 2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，验证了CASP 8AP 2基因作为HEK 293细胞中的工程靶标。缺乏CASP 8AP 2的HEK 293细胞显示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，而不会显着影响细胞生长和活力。转录组分析显示，细胞周期的失调，特别是细胞周期蛋白依赖性激酶抑制剂2A（CDKN 2A）基因的上调，有助于改善CASP 8AP 2缺陷细胞中的重组蛋白表达。结果验证CASP 8AP 2基因是用于在HEK 293细胞系中改善重组蛋白表达的可行工程化靶标。 除了蛋白质表达，哺乳动物细胞也是病毒表达的来源。因此，基于我们利用非编码RNA提高蛋白质产量的经验，我们希望鉴定与病毒结构蛋白和辅助蛋白共表达的miRNA或siRNA种类，以影响rAAV滴度的增加，而不依赖于所递送的转基因。我们期望病毒生产受益于不同的优化的miRNA谱。到目前为止，我们的工作一直是筛选条件的改进。筛选可以被认为是单独的转染/生产和转导/测定步骤。选择转染参数（平板接种密度、质粒浓度、转染试剂和反向和正向转染之间的天数）以优化（i）来自293 T/17细胞中ITR的荧光蛋白表达以及（ii）也通过荧光蛋白表达评估的在COS 7细胞中观察到的相应转导。互补地，选择转导参数以允许（i）在添加相同上清液批次的情况下的最高荧光信号，（ii）使液体处理误差最小化，和（iii）MOI/信号的水平显著低于非饱和。 相关参数的实例包括HEK 293 T/17细胞平板接种密度（3e 3）、总质粒量（0.12 g）、反向和正向转染试剂（Dharmafect 4和Viafect）以及用于评价转导的细胞系（COS 7）。基于最初的结果，改善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，转移到荧光素酶作为测定标记物，以及向筛选中添加新的特异性siRNA文库。该优化过程继续进行，并鉴定了影响高通量分析的更多参数。这项工作是与NCAT一起完成的。

项目成果

Coupling enrichment methods with proteomics for understanding and treating disease.

• DOI: 10.1002/prca.201400097
• 发表时间: 2015-02
• 期刊: PROTEOMICS CLINICAL APPLICATIONS
• 影响因子: 2
• 作者: Kumar, Amit;Baycin-Hizal, Deniz;Shiloach, Joseph;Bowen, Michael A.;Betenbaugh, Michael J.
• 通讯作者: Betenbaugh, Michael J.

Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells.

• DOI: 10.1371/journal.pone.0148075
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Chu C;Bottaro DP;Betenbaugh MJ;Shiloach J
• 通讯作者: Shiloach J

Large-scale screening identifies a novel microRNA, miR-15a-3p, which induces apoptosis in human cancer cell lines.

• DOI: 10.4161/rna.23339
• 发表时间: 2013-02-01
• 期刊: RNA biology
• 影响因子: 4.1
• 作者: Druz, Aliaksandr;Chen, Yu-Chi;Shiloach, Joseph
• 通讯作者: Shiloach, Joseph

Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1.

构建表达神经降压素受体 NTSR1 的重组 HEK293 细胞系。

• DOI: 10.1007/978-1-4939-2336-6_4
• 发表时间: 2015
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Xiao,Su;Shiloach,Joseph;Grisshammer,Reinhard
• 通讯作者: Grisshammer,Reinhard

Continuous production process of retroviral vector for adoptive T- cell therapy.

用于过继性 T 细胞治疗的逆转录病毒载体的连续生产过程。

• DOI: 10.1016/j.bej.2018.01.010
• 发表时间: 2018
• 期刊: Biochemical engineering journal
• 影响因子: 3.9
• 作者: Inwood,Sarah;Xu,Hui;Black,MaryA;Betenbaugh,MichaelJ;Feldman,Steven;Shiloach,Joseph
• 通讯作者: Shiloach,Joseph