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

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 γH2AX in an ITR-independent manner, however, canonical activation of p53α was uncoupled using rAAV-SynITR. Further hESC investigations revealed 2 additional novel findings: i) p53β is uniquely and constitutively active, and ii) rAAV infection, independent of the ITR sequence, induces activation of p53ψ. The data herein reveal an ITR-dependent rAAV transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering. ### Competing Interest Statement Support was provided by the UNC department of Ophthalmology and by the NIH (RO1AI072176-06A1, R.J.S/M.L.H). RJS and MLH are co-inventors of the SynITR platform which is licensed to AskBIO and RJS and MLH have received royalties. MLH is a cofounder (holds equity) of Bedrock and Astro Therapeutics for which he is also a consultant, in addition to Graybug Vision. RJS is the founder and a shareholder at Asklepios BioPharmaceuticals. He holds patents that have been licensed by UNC to Asklepios for which he receives royalties. He has consulted for Baxter Healthcare and has received payment for speaking. LS is an employee of Askleopios since April 2022. The additional authors declare no conflicts of interest.