LACK OF EXPRESSION FROM A RETROVIRAL VECTOR AFTER TRANSDUCTION OF MURINE HEMATOPOIETIC STEM-CELLS IS ASSOCIATED WITH METHYLATION IN-VIVO

We describe studies of gene transfer and expression of the human glucocerebrosidase cDNA by a Moloney murine leukemia virus (MoMuLV)-based retroviral vector in a murine gene transfer/bone marrow transplant (BMT) model. Pluripotent hematopoietic stem cells (HSCs) were assayed as the colony-forming units, spleen (CFU-S) generated after serial transplantation. Transcriptional expression from the MoMuLV long-terminal repeat (LTR) was detected at a high level in the primary (1-degrees) CFU-S and tissues of reconstituted BMT recipients. However, we observed transcriptional inactivity of the proviral MoMuLV-LTR in >90% of the secondary (2-degrees) CFU-S and in 100% of the tertiary (3) CFU-S examined. We have compared the methylation status of the provirus in the 1-degrees CFU-S, which show strong vector expression, to that of the transcriptionally inactive provirus in the 2-degrees and 3-degrees CFU-S by Southern blot analysis using the methylation-sensitive restriction enzyme Sma I. The studies demonstrated a 3- to 4-fold increase in methylation of the Sma I site in the proviral LTR of 2-degrees and 3-degrees CFU-S compared to the transcriptionally active 1-degrees CFU-S. These observations may have important implications for future clinical applications of retroviral-mediated gene transfer into HSCs, where persistent gene expression would be needed for an enduring therapeutic effect.