Recombinant Engineering Strategies for the Production of Therapeutic Coagulation Factor Proteins

Abstract

Dysregulated coagulation is a common clinical condition secondary to one of numerous blood dyscrasias and can be restored by therapeutic intervention with agents rich in coagulation factor proteins such as whole blood, fresh frozen plasma, prothrombin complex concentrates, and single coagulation factor concentrates. These agents are predominantly sourced from volunteer donors and consequently face supply, product uniformity, and pathogen contamination hurdles. These hurdles are a motivating factor behind the exploration of recombinant manufacturing alternatives that leverage the innate capabilities of a host organism combined with modifications to its genomic, transcriptomic, or proteomic profile to produce therapeutic proteins. Strategies for producing complex human proteins, especially coagulation factors, are highly homologous and consistently employ non-human mammalian cell lines and plasmid-based gene transfer techniques to engineer transgenic cell lines. This resource-intensive strategy must be carried out for each new protein of interest, a critical limitation for complex multi-protein cocktails, and can yield proteins with non-human post-translational modifications; a complication that can lead to reduced protein activity and immunogenic reactions. We sought to address these limitations by exploring a strategy in which a flexible transcriptional activation system could be applied to the expression of diverse single or multi-protein cocktails from human cells that are inherently capable of carrying out human post-translational modifications. Towards this goal, our pilot study investigated the Synergistic Activation Mediators (SAMs), a transcriptional activation system designed around the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease. Utilizing a plasmid-based strategy to express the CRISPR/Cas9-SAM system components our study validated the compatibility of the system with human cells, confirmed its programmability for mixed coagulation factor gene and protein expression, and optimized the SAM architecture for peak transcriptional activity. Our follow up study then took steps to engineer transgenic human cell lines stably expressing these optimized elements. Resulting transgenic cell lines were then programmed for expression of mixed coagulation factor gene and protein targets validating the flexibility of both the cells and the expression strategy. To expand the utility of our engineering approach we then replicated our engineering approach in a murine cell line, programmed it for expression of a murine specific coagulation factor protein, and validated subsequent gene overexpression. Our collective efforts establish proof-of-principle for a novel engineering strategy for the streamlined production of recombinant coagulation factor proteins with promise to address therapeutic gaps in the field of coagulation management. Further, the overall versatility of the SAM system including its trans-species compatibility extend its use beyond coagulation factor proteins to broad applications including therapeutic protein production, cellular network modulation and a multitude of basic, translational, and clinical areas of investigation.