Figure 6.

Summary of the vector-specific mechanisms of insertional mutagenesis and levels of vector genotoxicity in Cdkn2a^−/− mice. Specific culprits of insertional mutagenesis were predominant when specific lentiviral vector (LV) designs were used to treat Cdkn2a^−/− mice. For each vector design, we indicated the word cloud representation of the CIS gene identified and the major mechanism of insertional mutagenesis used with the relative median survival indicated into the brackets. In the word cloud representation (rectangles on the left), the most targeted genes are represented by larger letter size. The genotoxic potential of each vector varied from high (top), to moderate and low (bottom). The most genotoxic vectors were those capable of efficiently performing promoter insertion, such as LV.SF.LTR and SIN.LV.SF, leading to Braf activation (average of the median survival of the two groups is indicated). Vectors containing strong enhancer/promoters, such as SIN.LV.SF.GFP.PRE and SIN.LV.SF.PRE, efficiently performed enhancer-mediated activation of oncogenes as the main mechanism of insertional mutagenesis, leading to Map3k8 activation in our in vivo model (average of the median survival of the two groups is indicated). When SIN vectors contained moderate enhancer/promoters such as SIN.LV.PGK.GFP.PRE, both enhancer-mediated activation of oncogenes and gene inactivation are used as insertional mechanisms, promoting Map3k8 activation and inactivation of Pten and Rasa1 genes. Finally, when the activity of enhancers is shielded by the presence of insulator sequences, vector genotoxicity relies only on gene inactivation mechanisms.