title: The long terminal repeat negative control region is a critical element for insertional oncogenesis after gene transfer into hematopoietic progenitors with Moloney murine leukemia viral vectors.
abstract: Integrating vectors based on γ-retroviruses and containing full-length long terminal repeats (LTRs) have been associated with activation of oncogene expression and leukemogenesis in human gene therapy trials. Identification of the specific molecular elements of the LTRs that have a role in insertional oncogenesis events is important as it can lead to the development of safer gene transfer vectors. The negative control region (NCR) of the LTR is a particularly well-conserved sequence among mammalian γ-retroviruses with demonstrated regulatory activity of gene transcription in hematopoietic cells, which led us to hypothesize that this region may have a role in insertional oncogenesis after γ-retroviral vector (GV)-mediated gene transfer into hematopoietic progenitors. We used an in vitro assay of murine bone marrow cell immortalization to compare the immortalization capabilities of a series of GVs carrying murine leukemia virus (MLV) LTR deletion mutants. Compared with GV carrying the full-length MLV LTR, deletion of the complete LTR enhancer sequence showed significant reduction of immortalization rates. However, the use of a mutant LTR deleted of the enhancer sequence, with exception of the NCR, did not affect immortalization. Importantly, the inclusion of an LTR mutant devoid only of the NCR did show significant reduction of immortalization rates compared with the full LTR sequence. Therefore, our data point to the NCR as a key element for immortalization and justify additional studies to evaluate its specific role in MLV-mediated insertional oncogenesis.
authors.affiliation: Genetics and Molecular Biology Branch, National Human Genome Research Institute (NHGRI), Bethesda, MD, USA.|Department of Pediatrics, Kanazawa University Hospital, Kanazawa, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ikawa
authors.affiliation: Genetics and Molecular Biology Branch, National Human Genome Research Institute (NHGRI), Bethesda, MD, USA.|Department of Human Genetics, National Research Institute for Child Health and Development, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Uchiyama
authors.affiliation: Genetics and Molecular Biology Branch, National Human Genome Research Institute (NHGRI), Bethesda, MD, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Jagadeesh
authors.affiliation: Genetics and Molecular Biology Branch, National Human Genome Research Institute (NHGRI), Bethesda, MD, USA.|Division of Immunology and Allergy, University Hospital of Lausanne, Lausanne, Switzerland.
authors.forename: F
authors.initials: F
authors.lastname: Candotti
mesh_terms: D000818:Animals; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008979:Moloney murine leukemia virus / Q000235:genetics*; D016254:Mutagenesis, Insertional; D020079:Terminal Repeat Sequences