AAV Vector Transduction Restriction and Attenuated Toxicity in hESCs via a Rationally Designed Inverted Terminal Repeat

Abstract

Recombinant adeno-associated virus (rAAV) inverted terminal repeats (ITRs) induce p53-dependent apoptosis in human embryonic stem cells (hESCs). To interrogate this phenomenon, a rationally designed ITR (SynITR), deleted for p53 binding sites was evaluated for vector production and gene delivery. While SynITR genomes were decreased for transgenic genome replication compared to wtITRs, similar production titers indicated that replication is not rate-limiting. Packaged in the AAV2 capsid, wtITR and SynITR vectors demonstrated similar transduction efficiencies of human cell lines with no differences in reporter kinetics. Following rAAV2-wtITR infection of hESCs, rapid apoptosis was observed, in contrast, rAAV2-SynITR infection resulted in attenuated hESC toxicity with cells retaining their differentiation potential. While hESC particle entry and double stranded circular episomes was similar for the ITR contexts, reporter expression was significantly inhibited from transduced SynITR genomes. Infection of hESCs induced gamma H2AX in an ITR-independent manner, however, canonical activation of p53 alpha; was uncoupled using rAAV-SynITR. Further hESC investigations revealed 2 additional novel findings: i) p53 beta is uniquely and constitutively active, and ii) rAAV infection, independent of the ITR sequence, induces activation of p53 psi. The data herein reveal an ITR-dependent rAAV transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering.