Identification of genes related to spcific properties of mammalian cells

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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.