Our investigation using AAV vectors for the genetic modification of hESCs encountered an unexpected phenomenon: rAAV transduction directly correlated with hESC apoptosis. This phenotype was demonstrated to be independent of the viral capsid (, ), but rather, was attributed to the unique hESC DNA damage response elicited by the single-strand AAV origins of replication (ITR sequence), which are present on both wt and rAAV genomes. Consistently, DNA damage signaling cascades were induced upon rAAV transduction in hESCs and the apoptotic finale was dependent upon p53. The ITR dissection data from DNA microinjections supports a model in which G-rich repetitive elements of the AAV minimal origin of replication are the actual “apoptotic trigger”. The notion that this AAV “telomeric” DNA is toxic is consistent with reports of single-strand G-rich repetitive DNA at dysfunctional telomeres triggering a p53-dependent apoptotic response in other mammalian cell types 
. The inability of hESCs to tolerate transduced AAV genomes is the first example of rAAV toxicity in a wild type human cell and highlights the different DNA damage responses among hESCs, particular cancers 
, and differentiated cell types.
In contrast to reports of AAV-induced toxicity in p53 deficient cancer cells 
, the work herein demonstrates the opposite scenario in hESCs; rAAV-induced toxicity is dependent upon p53. This discrepancy likely reflects the unique role of p53 in hESCs in which DNA damage induces p53-ser15 phosphorylation, p53 accumulation and p53-dependent promoter trans-activation; however some downstream effectors are not elevated at the protein level 
. These observations were demonstrated in this work and the dramatic p53-depdendent induction of the p21 transcript upon AAV transduction did not allow detectable p21 protein. Consistently, work has demonstrated the post-transcriptional regulation of p21 protein abundance and that multiple micro RNAs, specifically expressed in pluripotent cells, target the 3′-UTR of the p21 message to down-regulate translation 
. Another notable attribute of p53 in hESCs is the ability to induce transcription-independent apoptosis via localization to the cytoplasm and direct activation of Bax followed by cytochrome C release and caspase activation 
. These reports are consistent with the hESC response to rAAV transduction described herein, in which transcriptional induction of known apoptotic effectors was not observed, however the event was p53-dependent and abrogated by caspase inhibitors.
Within the generalized term of pluripotency there appears to be various stages which can be delineated based on their response to DNA damaging agents, the significance of which is under current investigation. This notion is supported by hESCs of different origins which display distinct properties, such as levels of the Oct4 transcript and replication indices, which directly correlate to the induction of rapid apoptosis in response to rAAV transduction or etoposide (, S1
; unpublished data). This may not be surprising as hESCs exhibit high rates of apoptosis, undergo spontaneous differentiation and multiple passages induce genomic alterations 
. These observations are perhaps further illustrated by two groups who have recently reported the genetic modification of hESCs using rAAV 
. In the first instance, AAV gene correction was demonstrated in dissociated hESCs cultured in the presence of a ROCK inhibitor which has been found to pacify hESCs tendency towards apoptosis 
. The second instance is not readily explained, however, it is possible that a small fraction of hESCs demonstrate tolerance 
. We speculate that cell differences, perhaps due to variations in the initial aliquots or perhaps maintenance of pluripotency during culture, likely account for the observed differences. Intriguingly, hESCs and murine ESCs (mESCs) also demonstrate inherently different DNA damage responses, including the presence of a debatable G1/S checkpoint 
. In addition, mESCs undergo less spontaneous apoptosis and differentiation and are genomically stable in comparison to hESCs 
. Consistent with these reports, yet in contrast to the hESC transduction data, we, and others, have observed that mESCs tolerated AAV transduction, an (unpublished data; 
). A similarity is that by definition hESCs of different origins and mESCs are grouped under the general term of pluripotent, despite their altered response to DNA damage and predisposition for apoptosis.
Further characterization of the AAV induced DNA damage response in human pluripotent, cancer and differentiated cell types is currently underway and has strong implications for understanding the basis of oncogenesis and differentiation, as well as for the optimization of AAV as a DNA delivery vector in stem cells.