Open AccessLack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
Author(s) -
Pia-Maria Challita,
Donald B. Kohn
Publication year - 1994
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.91.7.2567
Subject(s) - biology , provirus , long terminal repeat , haematopoiesis , microbiology and biotechnology , viral vector , stem cell , transplantation , methylation , murine leukemia virus , gene expression , gene , genetic enhancement , dna methylation , virology , genetics , recombinant dna , medicine , surgery , genome
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.