Abstract

Engineering the Genome with the Sleeping Beauty Transposon System

Zoltan Ivics, Head of Division, Paul Ehrlich Institute

The Sleeping Beauty (SB) transposon system yields efficient stable gene transfer following gene delivery into the germline of several mammalian model species as well as into pluripotent and multipotent stem cells that are relevant targets for regenerative medicine and gene- and cell-based therapies of complex genetic diseases. The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We compared distributions of SB and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4+ T cells with respect to 40 chromatin states defined by combinations of genomic characteristics specified in 70 different genome-wide datasets. The SB transposon displayed the least deviation from random with respect to genome-wide distribution. We have identified remarkable parallels between integration site distributions of the PB transposon and the MLV retrovirus across all 40 chromatin states. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. Our data highlight the significance of vector choice to minimize the relative chance of insertional oncogenesis in clinical applications. We applied the SB system for non-viral gene delivery in both mouse and human hematopoietic stem cells. We optimized our protocols by applying minicircle vectors to deliver the transposon and synthetic mRNA to deliver the transposase into this hard-to-transfect cell type. With this optimized protocol in hand, we are in the process of setting up preclinical gene therapy in mouse models of Gaucher disease, an inherited lysosomal storage disorder. We have begun to dissect the molecular determinants of target site selection by the SB transposase. We have solved the crystal structure of the catalytic domain of the SB100X hyperactive transposase. The structure provides retrospective clues for some of