Song Liujiang, Hasegawa Tomoko, Brown Nolan J, Bower Jacquelyn J, Samulski Richard J, Hirsch Matthew L
Ophthalmology, University of North Carolina, 130 Mason Farm Rd, Chapel Hill, NC 27517, USA.
Carolina Eye Research Institute, 115 Mason Farm Rd, Chapel Hill, NC 27514, USA.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkaf013.
Adeno-associated virus (AAV) inverted terminal repeats (ITRs) induce p53-dependent apoptosis in human embryonic stem cells (hESCs). To interrogate this phenomenon, a synthetic ITR (SynITR), harboring substitutions in putative p53 binding sites was generated and evaluated for vector production and gene delivery. Replication of SynITR flanked transgenic genome was similar compared to wild type (wt) ITR, with a modest increase in vector titers. Packaged in the AAV2 capsid, wtITR and SynITR vectors demonstrated similar transduction efficiencies of human cells without toxicity. Following AAV2-wtITR vector infection of hESCs, rapid apoptosis was observed as reported. In contrast, infection by AAV2 vectors packaged with SynITRs attenuated the wtITR-induced hESC toxicity. While hESC particle entry and the abundance of double stranded circular episomes was similar for the ITR contexts, reporter expression was inhibited from transduced SynITR genomes. Mechanistically, infection of hESCs induced γH2AX in an ITR-independent manner, however, canonical activation of p53α was uncoupled using AAV-SynITR. Further investigations in hESCs revealed additional novel findings: (i) p53β is uniquely and constitutively active and (ii) AAV vector infection, independent of the ITR sequence, induces activation of p53ψ. The data herein reveal an ITR-dependent AAV vector transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering.
腺相关病毒（AAV）的反向末端重复序列（ITR）可在人胚胎干细胞（hESC）中诱导p53依赖性凋亡。为了探究这一现象，构建了一种在假定的p53结合位点存在替换的合成ITR（SynITR），并对其载体生产和基因递送进行了评估。与野生型（wt）ITR相比，侧翼为转基因基因组的SynITR的复制情况相似，载体滴度略有增加。包装在AAV2衣壳中的wtITR和SynITR载体在人细胞中表现出相似的转导效率且无毒性。如报道的那样，在hESC感染AAV2-wtITR载体后，观察到快速凋亡。相比之下，用SynITR包装的AAV2载体感染可减轻wtITR诱导的hESC毒性。虽然对于ITR情况，hESC颗粒进入和双链环状附加体的丰度相似，但转导的SynITR基因组的报告基因表达受到抑制。从机制上讲，hESC感染以ITR非依赖性方式诱导γH2AX，然而，使用AAV-SynITR可使p53α的经典激活解偶联。在hESC中的进一步研究揭示了其他新发现：（i）p53β具有独特的组成型活性，以及（ii）AAV载体感染，独立于ITR序列，可诱导p53ψ激活。本文的数据揭示了hESC特有的ITR依赖性AAV载体转导限制以及通过ITR工程对DNA损伤反应的操纵。
