Advanced Tools for Mammalian Genome Engineering

Recent advances in mammalian artificial chromosome design and engineering offer an alternative to existing methodologies for cellular bioengineering and address unmet needs to bioengineer more complex functionalities into human cells for subsequent commercialization. In this ST13B-001 application we propose to demonstrate utility of a novel chromosome-based gene delivery vehicle that is amenable to large genetic payloads while avoiding insertional mutagenesis and maintaining stable, long-term, gene expression. A cornerstone to our proposal is the utilization of a distinctive mammalian artificial chromosome technology termed Artificial Chromosome Expression System (ACE System), an autonomous chromosome-based circuit-board designed to contain approximately 70 site-specific recombination acceptor sites that can carry single or multiple copies of genes or DNA elements of interest. In Phase I of this solicitation we delivered a 144Kbp BAC containing the MCT1 genomic locus onto the platform ACE that had previously been loaded with a 168Kbp BAC, resulting in 312Kbp of total DNA added. In addition to demonstrating the specificity of integration we confirmed the structural stability of the genomic DNAs. In Phase II we will expand this disruptive technology by engineering additional robust and complex functionalities into the ACE system toward the goal of cell-based therapeutics.