Diverse genomic integration of a lentiviral vector developed for the treatment of Wiskott-Aldrich syndrome.

ABSTRACT

BACKGROUND: The genomic integration of a lentiviral vector developed for the treatment of Wiskott-Aldrich syndrome (WAS) was assessed by localizing the vector insertion sites (IS) in a murine model of gene therapy for the disease. METHODS: Transduced hematopoietic progenitor cells were transplanted into mice or cultured in vitro. The IS were determined in the genomic DNA from blood, the bone marrow of the animals and from cultured cells. RESULTS: Sequencing vector-genomic DNA junctions yielded more than 150 IS of which 50-70% were located in transcription units. To obtain additional sequences from the population of cultured cells, we used a vector-tag concatenation technique providing 190 additional IS. Altogether, the profiles confirmed the bias for integration in transcription units. The vector did not congregate as hotspots and did not appear to target specific categories of genes. The diversity of the IS reflected the initial marking of a polyclonal population of cells. However, relatively few vector IS were found in vivo because only 30-40 unique IS were identified in each mouse using this approach. Although four to ten IS were shared by the blood and bone marrow, no common IS was found between mice or between any mouse and the cultured cells. CONCLUSIONS: Taken as a whole, the pattern of genomic insertion of the WAS lentiviral vector was diverse and similar to that previously described for other HIV-1-derived lentiviral vectors. Testing cells destined for transplantation is unlikely to predict specific IS to be selected in vivo.