A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

TLDR

Mammalian artificial chromosomes (MACs) enable the autonomous, non‑integrating delivery of large genetic payloads, and the satellite‑DNA based ACE platform can be reproducibly generated de novo, purified, and applied to biopharmaceutical production, transgenesis, and gene‑based cell therapy. The authors established the ACE System to extend the utility of ACEs by providing a versatile platform for reliable engineering of artificial chromosomes. The ACE System comprises a Platform ACE with over 50 recombination acceptor sites that, together with targeting vectors and a site‑specific integrase, allows purified ACEs to be reintroduced and stably maintained in diverse recipient cell lines. The system enabled precise loading of one or two gene targets into LMTK(-) and CHO cells.

Abstract

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK(-) and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.