REMBAC - rapid efficient manifold baculovirus transduction for stable cell line generation

Efficient recombinant protein production depends on stable cell lines, especially for complex products like viruslike particles (VLPs) and adeno-associated viruses (AAVs) used in vaccines and gene therapy their use is aspirated. Conventional transfection methods often lead to inefficiencies and inconsistent product quality, making stable cell lines essential. However, developing these lines, particularly for large transgenes, is time-consuming and challenging. Current solutions, typically using Chinese hamster ovary (CHO) cells, may not meet the quality demands of VLPs and AAVs, which require human-like glycosylation. Therefore, a versatile, cell type-independent platform for stable cell line development is needed. Our system, based on baculoviral transduction of mammalian cells (BacMam), addresses this need. BacMam is cost-effective, scalable, and efficient, with key advantages: it doesn’t require high-biosafety labs, transduces various cell types, and integrates large DNA fragments into genomes. We developed the REMBAC platform (Rapid Efficient Manifold Baculovirus Transduction), enabling site-specific integration of large transgenes with customizable expression. This is particularly useful for cell-toxic proteins, and the system includes insulators to protect against host-cell silencing. REMBAC facilitates stable cell line development (SCLD) for a variety of biopharmaceutical applications, including VLP vaccines, AAV gene therapy vectors, and monoclonal antibodies. It combines BacMam’s versatility with homologous recombination for site-specific integration and uses the I-SceI homing endonuclease for precise transgene excision. A library of transfer vectors supports long-term, fine-tuned protein expression. This project aims to (i) optimize integration for model cell lines (HEK293 and HeLa) by adjusting homology region lengths, (ii) identify a genomic safe harbor (GSH) for HeLa cells, and (iii) characterize the transcriptional profiles of our plasmid toolbox. A major focus will be on genotypic and phenotypic characterization, verifying transgene integration, assessing copy number, checking for mutations or residual baculovirus sequences, and ensuring long-term cassette stability. We will also compare the growth and morphology of modified cells to wild-type cells to ensure the process does not negatively impact cell health. As proof of concept, we will compare REMBAC-based stable cell lines with conventional plasmid-based methods for producing influenza A VLPs and the therapeutic antibody Trastuzumab. We expect REMBAC to improve yield, consistency, and production time, demonstrating its broad potential for various biotechnological applications. Additionally, we plan to generate stable antigen-specific reporter cell lines using random genome integration. These reporter lines will simplify production by allowing easy identification and quantification of expression products and supporting potency testing during early production and clinical stages. In summary, this project aims to validate the REMBAC system’s efficiency and versatility for stable cell line development, optimize key components like homologous recombination sequences, and explore new genomic safe harbors. We aim to demonstrate REMBAC’s superiority over conventional methods in terms of efficiency and product quality while also providing valuable tools, such as stable reporter cell lines, for the biopharmaceutical industry.