Abstract
The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.
Highlights
Adeno-associated virus vectors based on serotype (AAV) 2 have shown great promise for therapeutic gene transfer when targeted to immune-privileged sites [1,2,3], but their efficacy has been modest when targeted to other tissues such as during hepatic gene transfer [4,5]
To study if AAV2 elicits an endoplasmic reticulum (ER) stress response, we first examined the major components of unfolded protein response (UPR) signalling pathways during AAV infection
While the basis for the differential induction of UPR between ssAAV2 and scAAV2 is not clear, it is possible that the UPR signalling pathways in response to scAAV infection may be regulated by tolllike receptor (TLR) family members
Summary
Adeno-associated virus vectors based on serotype (AAV) 2 have shown great promise for therapeutic gene transfer when targeted to immune-privileged sites [1,2,3], but their efficacy has been modest when targeted to other tissues such as during hepatic gene transfer [4,5]. Spliced XBP1 (sXBP1) protein is a potent transcription factor which translocates to nucleus to bind to UPR elements (UPREs) and activates many genes that are crucial for restoring cellular homeostasis [20] This highly regulated UPR response to ER stress reduces the demand on the protein-folding machinery and protects cells from further damage. In AAV2 mediated gene therapy, the concept of capsid protein dependent immunotoxicity is well documented [5,34] and several groups have shown that cellular cytoplasmic surveillance mechanisms such as the NF-kB signalling [35], MYD88 pathway or toll-like receptor (TLR-9) [36,37] signalling influence this process Since some of these pathways are directly influenced by UPR activation, we hypothesized that AAV2 infection induces ER stress and activates cellular UPR. Our studies were designed to comprehensively analyze the role of the three major UPR signalling arms in the life cycle of AAV vectors both in vitro and in vivo
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