Malignant Transformation of a PG13 Cell Line Producing an MFG Retroviral Vector Containing Wild-Type Murine Akt.

Abstract

Over-expression of the pro-survival Akt gene in rat mesenchymal stem cells (MSCs) by retroviral transduction has been shown to improve cardiac repair following MSC transplant into infarcted rat heart (Mangi et al., Nature Medicine, 2003), and may hold great potential for clinical myocardial repair following cardiac damage. In preliminary transplant studies in NOD-SCID mice, we transduced human MSCs with an MFG-based retroviral vector containing genes for wild-type murine Akt and green fluorescent protein (AIG). The PG13-AIG cells used to supply retrovirus were prepared by multiple rounds of transduction of NIH 3T3-derived PG13 retrovirus packaging cells with amphotropic AIG retrovirus produced by transient transfection of the AIG vector plasmid into ϕNX-A producer cells. Primary human MSCs were transduced with PG13-AIG supernatant, were expanded in vitro for 1-3 passages, and surgically implanted after adherence to ceramic cubes into NOD-SCID mice. Three weeks after implantation of 5x106 human MSCs, a tumor mass formed around 1 of 8 ceramic cube implants, characterized histologically as a sarcoma. Retroviral insertion site analysis by linear amplification-mediated (LAM)-PCR revealed multiple insertions present within the tumor DNA. Sequence analysis of several of these vector insertion sites revealed no homology to the human genome. However, two of the insertions gave highly significant sequence homology to mouse chromosomes 15 and 11, inside introns of the mouse Dxd17 putative RNA helicase gene and mouse hypothetical Gm252 gene, respectively. These results indicate that the sarcoma was formed in greater part by Akt-transduced mouse cells rather than by transduced human MSCs. A third insertion site exhibited highly significant sequence homology to the gibbon ape leukemia virus (GALV) envelope gene present within the mouse PG13 producer cells. As GALV-pseudotyped retroviruses produced by PG13 cells do not transduce mouse cells, this argues against the tumor resulting from Akt-transduced NOD-SCID mouse cells due to production of a replication-competent retrovirus by transduced MSCs. PCR analysis detected the presence of the herpes simplex virus thymidine kinase gene (a selectable marker present in PG13 producer cells) in the tumor DNA, which together with the insertion site analysis indicates that the sarcoma was formed at least in part by Akt-transduced PG13 fibroblast cells, which may have been a rare contaminant in the MSC cell implants due to carry-over of producer cells during PG13-Akt viral supernatant transduction of MSCs. No published studies have found a tumorigenic potential for the NIH 3T3-derived PG13 cell line, and transfection of wild-type Akt into NIH 3T3 cells has been shown in several studies to confer little or no transformation or tumor-formation capacity. Regardless of origin, cell transformation by retroviral transduction of wild-type Akt, possibly with insertional mutagenesis, is a potential risk for clinical use of Akt gene transfer for enhanced stem cell survival and repair.