87. Rationally Designed Inverted Terminal Repeats Enhance Transduction of Embryonic Stem Cells

Abstract

Despite the prevalence of AAV vectors in gene therapy, treatments combining AAV vectors and cell therapy have not progressed into the clinic despite previous reports of successful transduction of stem cells by AAV vectors. One potential explanation is AAV vector toxicity in stem and stem-like cells, as demonstrated in previous studies. In particular, the AAV inverted terminal repeat (ITR) sequence was shown to induce apoptosis in human embryonic stem cells (hESCs) in a p53 dependent manner. A bioinformatic analysis of putative transcription factor binding sites in the AAV2 ITR sequence revealed six putative p53 binding sites. A panel of rationally designed AAV2 ITRs was generated which abrogated putative p53 binding sites and demonstrated reduced p53 binding in an in vitro binding assay. The synthetic ITRs were capable of all of the vector production requirements and were verified by sequencing directly from vector preparations. AAV vector transduction and the hESC DNA damage response was altered by the synthetic ITRs. Importantly, our ITRs demonstrated reduced toxicity in hESCs compared to an AAV vector having the AAV2 ITR sequence. The collective results allude to modulation of the host's DNA damage response using rationally designed ITRs, which may enhance the relevancy of AAV vectors in stem cell therapies.