Gene transfer-based therapeutic approaches have greatly benefited from the 
ability of some viral vectors to efficiently integrate within the cell genome 
and ensure persistent transmission of newly acquired transgenes to the target 
cell progeny. However, integration of provirus has been associated with 
epigenetic repercussions that may influence the expression of both the transgene 
and cellular genes close to vector integration loci. The exploitation of genetic 
insulator elements may overcome both issues through their ability to act as 
barriers that limit transgene silencing and/or as enhancer-blockers preventing 
the activation of endogenous genes by the vector enhancer. We established 
quantitative plasmid-based assay systems to screen enhancer-blocker and barrier 
genetic elements. Short synthetic insulators that bind to nuclear factor-I 
protein family transcription factors were identified to exert both 
enhancer-blocker and barrier functions, and were compared to binding sites for 
the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors 
enclosing these insulator elements were produced at titers similar to their 
non-insulated counterparts and proved to be less genotoxic in an in vitro 
immortalization assay, yielding lower activation of Evi1 oncogene expression and 
reduced clonal expansion of bone marrow cells.