We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
734. The Influence of Vector Design on Integration Site Selection by gamma-Retroviral and Lentiviral Vectors.
- Authors
Recchia, Alessandra; Cattoglio, Claudia; Miccio, Annarita; Antonelli, Antonella; Testa, Anna; Facchini, Giulia; Sartori, Daniela; Ferrari, Giuliana; Mavilio, Fulvio
- Abstract
Integration of gamma-retroviral and lentiviral vectors follows non-random patterns in mammalian genomes, with a shared preference for active chromatin regions but a different propensity to integrate in the proximity of transcription start sites. Retroviral integration preferences increase the risk of activation and/or deregulation of genes surrounding the integration site, and account for a concrete risk of insertional mutagenesis/oncogenesis. The molecular basis of the preference of retroviral pre-integration complexes is poorly understood, particularly in the case of gamma-retroviruses. Here we investigate the effect of vector design on the integration pattern of MLV- and HIV-derived retroviral vectors in the context of human CD34+ hematopoietic stem/progenitor cells. In both vector frameworks, we have compared three different designs, sharing an identical vector backbone (a GFP gene under the control of viral LTRs and a ?LNGFR reporter gene under the control of an internal promoter) but differing in the LTR configurations. The first design maintains wild type LTRs, the second carries a deletion in the U3 region (SIN), while in the third the U3 enhancer is replaced by an erythroid-specific enhancer element of the GATA-1 gene. Finally, the MLV U3 enhancer was swapped into an HIV LTR in a lentiviral vector context. A linker-mediated PCR technique (LM-PCR) has been optimized to evaluate the integration patterns of the three different constructs in CD34+ cells. 200 to 400 integration events were collected for each vector and mapped onto the human genome. Integration sites were comparatively analyzed, and correlated with the activity of genes located within an interval of 30 kb upstream and downstream from the insertion sites, as estimated from microarray gene expression profiles of mock-transduced CD34+ cells. Genomic sequences flanking the integration sites (±500 bp) were analyzed by a series of parameters, including a principal-component analysis of the content and arrangement of putative transcription factor binding sites. Our data indicate that LTR modification affects the normal retroviral integration pattern, suggesting that vector design may have an impact on the safety characteristics of retroviral vectors in clinical applications.Molecular Therapy (2006) 13, S284–S284; doi: 10.1016/j.ymthe.2006.08.815
- Subjects
ONCOGENIC viruses; RNA viruses; GENE expression; HUMAN gene mapping; GENETIC regulation; GENOMES
- Publication
Molecular Therapy, 2006, Vol 13, pS284
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.815