Utilizing CRISPR/Cas9 to identify chromosomal loci

Current methods for imaging transgene integration sites use DNA-FISH, which is a time- and labor-intensive process. Every genomic target requires a costly probe specific to the locus of interest. Through CasFISH, the only customized reagent needed is a target-specific crRNA, while a singular tracrRNA, dCas9, and antibody set can be applied to any newly synthesized crRNA, significantly reducing cost and effort when targeting multiple sites. The CRISPR/Cas9 system provides highly specific targeting of genomic DNA by means of a Cas9 nuclease complexed with guide-RNA which binds a 20 base-pair target sequence within the genome. Introducing two point-mutations to the catalytic region of Cas9 results in a nuclease-inactive enzyme referred to as dCas9. This catalytically “dead” system can be used by leveraging the intact DNA-interrogative properties to direct proteins to areas of interest within the genome. Our group has applied this approach to image telomere-specific repeats on chromosomes in metaphase spreads via immunofluorescence within our host cell line, CHOZN GS-/-. Utilizing the CasFISH method has been successful for us and others in targeting large, repetitive regions of interest, but to our knowledge, there has been no success in targeting smaller, non-repetitive regions using CasFISH alone. Our current work seeks to solve this problem by combining CasFISH with other existing technologies, such as DuoLink PLA (proximity ligation assay) or quantum dots, which are artificial semiconductor nanoparticles. Here, we detail our efforts in exploring these technologies, which promise to reduce the time and effort spent in visualizing transgene integration sites and other genomic loci of interes

Development of a platform expression system using targeted integration in Chinese Hamster Ovary cells

In recent years the biomanufacturing industry has seen significant improvements in recombinant protein production titers due to advancements in protein expression technologies as well as media, feed and manufacturing process development. However, the standard methods of recombinant cell line development have remained relatively unchanged. The majority of the biopharma industry introduces transgenes into Chinese Hamster Ovary (CHO) cells using mechanical or chemical transfection processes followed by metabolic or antibiotic selection of stable recombinant pools. Through this process, the transgene(s) are randomly integrated into the genome, often times resulting in significant heterogeneity within the stable pools. Individual recombinant CHO cells within the pools can vary greatly in their growth and productivity profiles, product quality attributes, and genetic stability. To isolate and identify the best performers, the time and resource consuming process of single cell clone generation and characterization is used, commonly requiring hundreds to thousands of clones to be characterized to find those suitable for manufacturing processes. In contrast, the use of targeted gene integration in cell line development programs will shorten timelines and reduce the burden of clone screening and characterization. The ability to integrate transgenes at a well-characterized and stable site will decrease heterogeneity in stable pools and lead to more consistent clone performance and product quality. Targeted Integration can also enable researchers to perform specific modifications of glycosylation and metabolic pathways but this abstract will focus more on improving cell line development and manufacturing applications. In this abstract we describe our strategy for developing a CHO expression platform that enables targeted and site specific integration of transgenes. We have generated clonal cell lines in which a landing pad has been randomly integrated into the CHOZN® genome at a low copy number. The landing pad contains a recombinant IgG expression cassette enabling us to screen for clones that support high and stable recombinant protein expression. Following this approach, we have identified and characterized several high expressing landing pad clones with performance characteristics suitable for commercial manufacturing processes. To bring these clones to the CHO industry we must first remove our IgG cassette from the landing pad and exchange it with a regulatory friendly and easy to screen GFP reporter. The landing pad was designed with Lox sites flanking the IgG cassette so that it could be excised using Cre Recombinase Mediated Cassette Exchange (RMCE). The landing pad itself remains integrated in the genome and acts as a placeholder for future site specific integration. Following another round of single cell cloning and characterization we can ensure that the original IgG cassette was cleared from the CHO genome and only the GFP reporter is expressed at the landing pad site. Our team is currently testing all of the top clones that have been derived from this process to select a single clone that will be taken forward for commercialization. We are hoping that upstream biopharma teams will soon be able to integrate their clinical molecules into a landing pad site available in our CHO cell line. The ability to integrate a transgene at a single genomic locus should decrease off target effects and increase the homogeneity of the resulting pools, thus reducing the burden and time required for clone screening and stability studies. Biopharma teams using such a platform expression system will be able to generate more high producing clones with fewer resources and get pipeline molecules to clinic more quickly

Genetic engineering of MMV virus resistance into CHO cells: Probing the role of various CHO sialyltransferases in virus binding and internalization

Contamination by the parvovirus Mouse Minute Virus (MMV) remains a continuing challenge in CHO biopharmaceutical production processes. As part of developing a risk mitigation strategy against such events our group has evaluated the genetic engineering of Chinese Hamster Ovary (CHO) cell lines to create a new host cell line that would be resistant to MMV infection e.g by inhibiting viral attachment to a cell surface receptor. While the exact functional receptor for MMV binding to CHO cell surface is unknown, previous work in our group has validated the role of sialic acid on the cell surface as important for cell surface binding and internalization of the MMV virus. Sialyltransferases are a group of enzymes that catalyze the transfer of sialic acid to the glycan moiety of glyoconjugates. In-vitro glycan-arrays have indicated that MMV binds preferentially to α-2,3 linked sialylated glycans and do not bind to α-2,6 sialylated glycans. CHO cells have six different sialyl transferases (ST3Gal 1-6), that transfer sialic acid in a α-2,3 linkage specific manner. In this work we systematically knocked out these genes and then probed for their role in MMV infectivity by challenging each cell line for their ability to resist viral entry. Results showed that St3Gal4 had a predominant effect on MMV infectivity with functional deletion resulting in a 54-88% decrease in infection compared to the Control WT cells. Additional deletion of St3Gal6 had little to no additional benefit in terms of viral resistance. Interestingly, the St3Gal4 and the St3Gal4 + 6 double mutants displayed both a decrease in viral binding to the cell surface as well as viral internalization and replication. Gene knock out of another sialyltransferase of the same family St3Gal 3 had a wide range of effect on MMV infectivity (18-84% of WT), which could be explained by a non-specific clonal effect. Based on the high resistance profile of cells with truncated O-glycosylation (COSMC knock-out clones), we hypothesized that the sialyltransferase St3Gal1 would have a significant effect on MMV infectivity, which could be further enhanced by combining it with an St3Gal4 deletion . We also sought to replace the α-2,3 sialylated phenotype with α-2,6 sialylation on the glycoproteins expressed in the viral resistant host cell lines by over-expression of the St6Gal1 gene. Such an approach would also make the therapeutic protein have more “human” like glycosylation. Model recombinant proteins were transfected into the new host cell lines and growth, IgG productivity and product quality studied. Our data demonstrate that viral resistance against MMV virus can be incorporated into CHO production cell lines, adding another level of “defense”, against the devastating financial consequences of this virus infection, without compromising recombinant protein yield or quality

A production-adapted, multi-auxotrophic, CHO cell line for reducing monoclonal and bispecific antibody cell line development timelines

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Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS−/− cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients