476. Human Mesenchymal Stem Cells Genetically Modified To Express High Levels of Erythropoietin Through Transfer of an Artificial Chromosome Engineered Using the ACE System

The ACE System is a versatile, reliable system for genetically modifying cell therapies, generating transgenic animals, and engineering mammalian cells for high expression of a recombinant protein. Key components of the ACE System include an artificial chromosome (Platform ACE) encoding multiple (>50) DNA site-specific integration sites (acceptor sites); a targeting vector (ACE Targeting Vector) encoding both a Platform ACE-specific DNA donor site and the therapeutic gene of interest; and a proprietary DNA recombinase (ACE Integrase) that catalyzes the site-specific recombination of ACE Targeting Vectors onto the Platform ACE. ACEs are a promising means of genetically modifying and engineering cells for cell therapy, as they are stably maintained, autonomous, non-integrating, easily purified by flow cytometry (de Jong et al, 1999) and readily transfected into a variety of cell types, including human adult stem cells (deJong et al, 2001; Vanderbyl et al, 2001; Vanderbyl et al, Stem Cells in Press).In this study, we engineered a Platform ACE to encode both the humanized renilla green fluorescent protein (hrGFP) and the human erythropoietin (epo) genes. A Platform ACE, carried in a CHO cell line, was first loaded with an ACE Targeting Vector encoding the hrGFP gene. Systematic analysis of drug resistant colonies by PCR, FISH and flow cytometry demonstrated that greater than 80% of the analyzed clones contained hrGFP-loaded ACEs and expressed GFP. Subsequent loading of the hrGFP-ACE with a second ACE Targeting Vector encoding the human epo gene yielded epo-loaded and expressing ACEs in approximately 83% of the GFP+ colonies. The resultant hrGFP-epo-ACEs were isolated and purified by flow cytometry and transfected into adult human mesenchymal stem cells (hMSCs) using cationic agents. The transfected hMSC population, which was enriched to 20% GFP+ cells by flow cytometry, secreted epo in the range of 50-100 IU/10 6 cells/day. These levels of epo expression are comparable to those attained with multiple cycles of epo-retroviral transductions in MSCs (Bartholomew et al, 2001).Currently we are implanting epo-expressing hMSCs into NOD/SCID mice and will monitor hematocrits over extended periods of time. We believe the above study will provide seminal data demonstrating that artificial chromosomes engineered using the ACE System will provide a safe and viable approach for ex vivo gene-modified cell therapies.