Hierarchical DNA assembly for advanced applications in biopharmaceuticals production and cell therapy

This project proposal is a joint initiative of one industrial (Lek) and three academic partners (IJS, NIB and FCCT). The project group is proposing a project in development and improvement of hierarchical DNA assembly for advanced applications in biopharmaceuticals production and cell therapy. Lek is a lead pharmaceutical company in Slovenia that develops cell lines to produce biopharmaceuticals, and NIB focuses on the development of advanced cell therapies for cancer and autoimmune diseases. Both institutions are facing similar challenges in their research projects, which is assembly of complex DNA molecules encoding genes for multiple proteins. Although DNA synthesis costs dropped significantly in the past decade, it is currently not possible to order an advanced molecular cloning service on the market. Laboratories must therefore rely on their own capabilities for the assembly of synthetic genes into DNA constructs that can be delivered into biological systems, which can be time-consuming and expensive. Therefore, Lek and NIB identified two key collaboration academic institutions (FCCT & JSI) both having substantial knowledge and capabilities in solving this challenge. Production of biopharmaceuticals requires testing the effect of multiple genes, promoters, shRNAs, and other features that affect the production and/or stability of the production cell lines before products reach the quality required for production. Also for cell therapy, many different immune receptors and accessory molecules are tested in order to improve effectiveness and safety of engineered cells. In this proposal, our overarching goal is to develop a hierarchical DNA assembly platform for advanced applications in biopharmaceutical production and cell therapy. We will achieve this aim with three objectives: (1) Develop a DNA assembly platform, (2) Validate and improve generated construct in mammalian cells, and (3) Demonstrate the utility of the platform by two distinct biomedical implementations. The design of immune receptors emerged as one of the critical factors in developing efficacious cellular products for the immunotherapy of cancer and autoimmune diseases. We will first utilize the hierarchical DNA assembly platform to assemble a complex genetic construct that comprises an immune receptor, an accessory molecule, and a selection marker. Next, we will utilize a shRNA-enabled hierarchical DNA assembly platform to downregulate T-cell receptor (TCR) expression. Together, this will demonstrate how our proposed molecular cloning platform can be utilized to (1) generate an advanced genetic construct for cell therapy and (2) provide a unique solution to one of the outstanding challenges in engineered T cell therapies, which is the downregulation of endogenous TCR surface expression. In vitro methods for DNA assembly are the cornerstone of genetic engineering and have been well developed. However, assembly of large DNA fragments and especially fast and efficient combinations of such elements is far from trivial. We will combine in vitro techniques with an in vivo DNA assembly relying on homologous recombination in yeast Saccharomyces cerevisiae cells. This approach, named hierarchical DNA assembly is not entirely new, but its potential implications have not been fully investigated. Based on our previous results and expertise we are confident that hierarchical DNA assembly is the way to solve the challenges by which Lek and NIB have been confronted. The project is expected to generate a patentable technology platform for hierarchical DNA assembly. Commercialization of the technology platform is foreseen within a newly established spin-out company, which will provide capacities for customary DNA assemblies to Lek and other customers.