Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo (original) (raw)

Abstract

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 degree) 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 degrees) CFU-S examined. We have compared the methylation status of the provirus in the 1 degree 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 degree 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.

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