title: Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
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.
authors.affiliation: Department of Microbiology, University of Southern California School of Medicine, Los Angeles.
authors.forename: P M
authors.initials: PM
authors.lastname: Challita
authors.forename: D B
authors.initials: DB
authors.lastname: Kohn
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D005962:Glucosylceramidase / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008979:Moloney murine leukemia virus / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D010948:Viral Plaque Assay
chemical_list: C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D005962:Glucosylceramidase