1. ¹Preclinical Programs, Chromos Molecular Systems, Inc., Burnaby, BC, Canada
2. ²Pathology and Internal Medicine, The Ohio State University, Columbus, OH
3. ³Center for Stem Cell and Regenerative Medicine, Cleveland, OH

Abstract

The ACE System is a potent biological engineering system consisting of a mammalian artificial chromosome engineered with multiple site-specific integration sites, expression-optimized shuttle vectors to specifically transfer genes, and a proprietary integrase to catalyze specific incorporation of a payload onto the ACE. ACEs are promising gene delivery vehicles for gene-based cell therapies as they are stably maintained, non-integrating, autonomously replicating, and are easily isolated to high purities by flow sorting. We published the first reports of the transfer and stable transgene expression of a mammalian artificial chromosome into hMSCs and hHSCs (Stem Cells 22:324|[ndash]|33, 2004; Exp Hematol 33:1470|[ndash]|1476, 2005). We will update our progress, including an hMSCs enrichment strategy resulting in stable EPO transgene expression, more than 50 days post EPO-ACE transfer, at levels of 100|[ndash]|200 IU/cell/day.

We also report the first successful ACE transfer into human embryonic stem cells (hESCs). We quantified the delivery of IdUrd- labeled ACEs to hESCs, via flow cytometry at 24|[ndash]|48 hours post- transfection, a screening technique that utilizes a FITC-conjugated anti-BrdUrd B44 clone antibody that binds to the IdUrd-DNA adduct. We detected IdUrd-labeled ACEs in 13% of the cells 24|[ndash]|48 hours post-transfer. Gene expression studies are underway. The combination of ACEs and the multipotency of adult & embryonic stem cells represent a unique approach for the study of stem cell fate/biology and for the development of novel gene-based cell therapies.