2. Identification and Ranking of Different Chromatin Insulators to Block Vector-Driven Enhancer-Mediated Insertional Mutagenesis In Vivo

Aimed at refining the safety profile of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications we investigated the impact of chromatin insulators (CI) on vector-mediated genotoxicity. Specifically, we studied four recently identified CI whose function is mediated by CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates, and cloned these CI in the LTRs of a SIN. LV with a strong enhancer/promoter in internal position (CI. SIN. LV).We took advantage of two sensitive in vivo genotoxicity assays based on the systemic injection of LVs in newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice, that allow to measure vector-induced genotoxicity as accelerated tumor onset proportional to the genotoxic potential of the tested LV.CI. SIN. LVs displayed slightly not statistically significant improvement in the median survival time vs. the uninsulated SIN. LV counterpart (ranging from 193.5 to 214 days vs. 186 days, respectively) in Cdkn2a−/− mice.In Cdkn2a+/− mice, two insulated vectors significantly improved the median survival time, which resulted non-statistically different from Mock mice (450 and 511 vs. 505.5 days respectively), while the other two CI. SIN. LVs studied resulted to be still slightly genotoxic (median survival time: 412 and 429.5 days). To gain more insights on the safety profile of these LVs we retrieved and analyzed the vector integration sites (IS) (n>14000 IS) and identified common integration sites (CIS) in the murine tumors generated in our experimental framework and in both murine models.In Cdkn2a−/− mice, uninsulated SIN. LV-induced tumors harbored activating integrations targeting Map3k8 oncogene, while tumors obtained in mice treated with two out of four different insulated LVs significantly reduced the frequency of tumors with Map3k8-activating insertions. The reduced targeting frequency of Map3k8 was accompanied by a skewing of integrations inactivating Pten, Rasa1 or other tumor-suppressors, an escape genotoxicity mechanism on which insulators cannot act.In Cdkn2a−/+ mice we identified different predominant CIS genes targeted by the different insulated vectors. These data show that heterozygous Cdkn2a−/+ mice allow discriminating between more subtle shades of genotoxicity of the different vector designs and are therefore instrumental to understand the different molecular mechanisms of insertional mutagenesis and ways to avoid them. Interestingly by comparing the results from both in vivo assays we observed that one CI displayed superior safety profile in terms of significant improvement in the median survival time and/or in terms of reduced oncogenic CIS identified.In summary we validated new human-origin insulator elements able to block SIN. LV genotoxicity in vivo. Overall, these data highlight the importance of stringent in vivo genotoxicity testing of improved vector versions and support the use of CI for future gene therapy applications.