Orthogonal Transposons for Iterative Genome Engineering of Mammalian Cells

Abstract The contemporary shift toward multispecific antibodies, antibody-drug conjugates (ADCs), and bespoke glycoengineered therapeutics have exposed the limitations of standard genomic engineering tools. This paper presents a novel iterative engineering paradigm utilizing the Leap-In Transposase® platform. By leveraging a suite of three mutually orthogonal transposase-transposon systems, we demonstrate the sequential modification of the Chinese Hamster Ovary (CHO) genome to achieve three distinct functional outcomes: (i) First, the creation of a glutamine synthetase (GS)-deficient host (CHO-K1-GS) via targeted knockdown, (ii) Second, the integration of multiple copies of a model therapeutic IgG1 for expression, and (iii) Third, the subsequent knockdown of the fucosylation pathway to modulate the glycan profile of the expressed IgG1. Genetic stability (copy number & sequence) of each integration event was confirmed using Targeted Locus Amplification (TLA) and Next-Generation Sequencing (NGS). Functional stability (expression levels, metabolic phenotype, and glycan phenotypes) was confirmed using standard cell culture and analytical techniques. Crucially, the truly orthogonal nature of the transposase-transposon pairs prevents cross-mobilization and ensures the structural and functional integrity of previously integrated cargo. This study establishes a “What You See Is What You Get” (WYSIWYG) methodology that provides a robust, scalable, and predictable framework for developing next-generation complex biopharmaceutical manufacturing cell lines. Competing Interest Statement Maggie Lee, Jeremy Minshull, Varsha Sitaraman, and Ferenc Boldog have filed patent applications related to the subject matter of this manuscript.