Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.

AUTOR(ES)

Challita, P M

RESUMO

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.

Documentos Relacionados

• Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells
• Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.
• Steel factor influences the distribution and activity of murine hematopoietic stem cells in vivo.
• Retrovirus-mediated gene transfer of human adenosine deaminase: expression of functional enzyme in murine hematopoietic stem cells in vivo.
• Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.