Modulation of AAV transduction and integration targeting by topoisomerase poisons

Adeno-associated virus (AAV) is a widely used vehicle for gene delivery, lending interest to developing methods for enhancing AAV transduction and transgene expression. Here, we profile the function of several topoisomerase poisons, which are small molecules that stabilize topoisomerase enzymatic intermediates, where topoisomerase enzymes are covalently bound at chromosomal DNA breaks. As previously observed, we found that the topoisomerase poisons camptothecin (CPT), doxorubicin (DOX), and etoposide (ETO) increased AAV transduction in cultured cell models. DOX and ETO, small molecules that specifically inhibit type II topoisomerases and so stabilize double-strand breaks, were found to boost integration of AAV DNA into the host cell chromosome. Analysis of integration site distributions showed that integration targeting was altered, so that integration in the presence of DOX or ETO was favored near actively transcribed regions. Locations of topoisomerase II binding sites were inferred from genomic data using a novel machine learning platform, and integration in the presence of DOX or ETO was found to be selectively favored near inferred topoisomerase II binding sites. These data help guide development of improved transduction protocols using these reagents and establish that DOX and ETO can control AAV integration targeting. [Display omitted] Co-delivery of type I and type II topoisomerase poisons is known to increase AAV transduction but impact on vector integration is unknown. Bushman and colleagues found that type II topoisomerase poison-treated cells have higher integration frequencies and more integration targeting to active genes and type II topoisomerase sites predicted by machine learning.